All projects in the institute pertain to one of the following 3 categories:
- Identification, characterization and optimization of new TB drug leads
- Development of new tools to facilitate TB drug discovery
- Identification of the molecular targets of and/or mechanisms of resistance to anti-TB agents
TB drug discovery efforts include high throughput screening of synthetic compounds and natural products against both replicating and non-replicating Mycobacterium tuberculosis conducted within a biosafety level 3 laboratory. Leads are also derived from the bioassay-guided isolation of natural products from microorganisms, marine invertebrates and higher plants.
Complementing whole cell-based screening efforts are target-based projects, in some cases with in silico modeling components. All have a functional screening component and all target-based projects are performed as collaborations with faculty in the College of Pharmacy specializing in computational chemistry, medicinal chemistry and structural biology or with collaborators outside of UIC. Other projects are examining genes which may be essential for virulence or latency to assess their potential as drug targets.
Lead identification/optimization projects consist of bioassay support for chemists outside of UIC. The remaining projects involve medicinal chemistry that is performed within the Institute. Besides assessment of anti-TB activity in vitro and in vivo, lead optimization efforts include drug metabolism/PK studies and pharmacophore ID/QSAR analyses.
Finally the ITR is a leader worldwide in the development of new HTS-compatible assays to detect new compounds active against both replicating and non-replicating M. tuberculosis.
Lead identification begins with the identification of a compound or class of compounds (or extracts in the case of natural products) with the ability to inhibit or kill Mycobacterium tuberculosis while being less toxic or non-toxic for mammalian cells. The process involves multiple steps to assess potency, selective toxicity, anti-microbial spectrum of activity, and stability in the presence of gastric acid and liver enzymes. Compounds with favorable profiles proceed onto evaluations of pharmacokinetics (measuring concentrations in the blood and in the lungs) and the ability to inhibit/kill M. tuberculosis in a living host. A compound that can do the latter is considered to be a lead and efforts are then made to optimize its safety and efficacy through chemical modification.
We have completed the screening of over 200,000 actinomycete fermention extracts from the Extract Collection of Useful Microorganisms (ECUM), at Myongji University in South Korea. In this collaboration ECUM performs fermentations and primary extractions and and the ITR does both the natural products chemistry as well as all of the biology. A small number of extracts were prioritized based on potency, selective toxicity, spectrum of activity and lack of cross resistance with TB strains resistant to rifamycins and aminoglycosides. Large scale re-fermentations have provided sufficient biomass to allow the isolation of a number of active principles with MICs below 1 ug/ml. This project makes extensive use of high speed countercurrent chromatography using new techniques developed in the laboratory of Dr. Guido Pauli, an affiliate faculty member. Ecumicin, a novel cyclic peptide with activity in TB-infected mice, was discovered in this program and is currently the subject of efforts to improve oral bioavailability. Our most recent efforts involve the targeted search for minor constituents, the development of high throughput co-culture and expansion of our target panel to include the so-called ESKAPE pathogen panel.
Another major collaborative effort is with the Murphy Lab, also in the College of Pharmacy at UIC. This project focuses on novel actinomycetes cultured from fresh and saltwater sediments.
Our primary collaboration for examining fungal metabolites is with Fungi Perfecti, founded by the well-known mycologist and visionary Paul Stamets. We are focusing on species of mushrooms used for treating TB centuries ago and we perform the natural products chemistry as well as the biological profiling. See the search for one such species in the Pacific Northwest.
Fructose 1,6 bisphosphatase is a gluconeogenic pathway enzyme that was identified as being essential for persistence in a mouse model of TB infection by Dr. Farah Movahedzadeh’s lab in the ITR. Working with Dr. Cele Abad-Zapatero, a crystal structure has been obtained. Current efforts are focused on identifying inhibitors of the enzyme.
clpC1 is an essential chaperone protein in M. tuberculosis which is involved in protein degradation and is the target of the natural products cyclomarin, ecumicin and rufomycin. We isolated the latter two compounds from actinomycete fermentation broths and have generated and mapped resistant mutants. We are studying the interaction of the cyclic peptides with the target by surface plasmon resonance, co-crystallization and functional inhibition, the latter with colleagues at Myongji University and Harvard University.
- We have previously developed high throughput compatible assays for determining inhibition of killing of both replicating and non-replicating cultures of M. tuberculosis. This includes the development of recombinant strains of M. tuberculosis expressing fluorescent or luciferase proteins that are used in rapidly assessing activity of candidate drugs. We conduct this assay in our internal projects and for our external collaborations. Current efforts are exploring the use of new fluorescent reporter proteins such as mCherry.
- We are developing a bioautography methods that employs an avirulent strain of M. tuberculosis equipped with a luxABCDE reporter. Using extracts or fractions this method can detect anti-TB compounds following their seperation on high performance TLC plates. Such compounds can be de-replicated by obtaining HR-MS data from the corresponding replicate plates using a TLC-MS interface device. Ultimately our goal is to obtain NMR data from prep samples, allowing for the acquisition of structural data, MICs and selectivity indices without the requirement of going through the classical bioassay-guided isolation process.