Positron emission tomography (PET) radioisotopes, such as carbon-11 (11C) and fluorine-18 (18F), will be incorporated into current antibiotics, such as the tuberculosis drugs isoniazid and rifampicin, in order to directly identify the protein target(s) for these molecules in living cells. Separation methods will be employed that that preserve non-covalent complexes, and PET labels will be incorporated without altering the structure of the drug. This approach thus represents a dramatic improvement on existing methods, in which drug molecules are covalently modified in order to create affinity matrices or photoaffinity labels, and will provide critical information for rational drug design. The use of PET radioisotopes has several advantages compared to methods that relay on conventional radiolabels (14C and 3H), such as dramatic improvements in sensitivity, ease of protein identification and the ability to image drugs in vivo. Importantly, the introduction of PET labels into pathogen-specific biomolecules will facilitate the use of PET to detect and localize pathogens in infected patients. The initial focus will be on pathogens such as Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus, however the approach has general applicability.
Aim 1 will identify the protein target(s) of the front-line tuberculosis drugs rifampicin and isoniazid as well as the target(s) of novel microbial fatty acid biosynthesis inhibitors. Synthetic methods will be developed to rapidly incorporate 11C or 18F labels into the drugs without altering the overall structure of each molecule. Bacteria will then be exposed to labeled drugs and fractionated under non-denaturing conditions using methods such as size-exclusion chromatography and isoelectric focusing with in-line radiation detection to identify fractions containing protein-drug complexes. After allowing the samples to decay, protein-drug targets will be identified using standard proteomic methods.
In Aim 2 the labeled compounds developed in Aim 1 will be used to image the tissue distribution and metabolic profile of the drugs in healthy control animals using PET.
This Aim will evaluate the effect of different routes of drug administration on drug bioavailability to target organs such as the lung. Ultimately, the imaging studies will be extended to animal models of infection and infected patients.
There is a continuous need to develop new drugs that are effective against pathogens such as drug- resistant Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus. However, the rational development of new drugs is hindered by the lack of sophisticated methods for determining how drugs work ij living systems. The objective of the current proposal is to develop a method in which the protein targets of drugs in cells can be directly determined. This method relies on the incorporation of short-lived radioisotopes that decay to emit body-penetrating photons, and thus the reagents that are developed should also be useful for directly detecting pathogens in infected patients.
| Weinstein, E A; Liu, L; Ordonez, A A et al. (2012) Noninvasive determination of 2-[18F]-fluoroisonicotinic acid hydrazide pharmacokinetics by positron emission tomography in Mycobacterium tuberculosis-infected mice. Antimicrob Agents Chemother 56:6284-90 |
| Liu, Li; Xu, Youwen; Shea, Colleen et al. (2010) Radiosynthesis and bioimaging of the tuberculosis chemotherapeutics isoniazid, rifampicin and pyrazinamide in baboons. J Med Chem 53:2882-91 |
