Tuberculosis (TB), an infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb), kills nearly 2 million people a year and is a major cause of morbidity and mortality worldwide. The increasing prevalence of multi-drug resistant (MDR) strains that fail to respond to all currently available drugs underscores the critical need for new therapeutic strategies. DNA topoisomerases (topos) are a class of enzymes responsible for maintaining the correct topological state of DNA molecules by modulating the degree of supercoiling through the breakage and reforming of covalent phosphodiester bonds in the DNA backbone. With the precedent-setting success of several anticancer and antibacterial drugs that target topos, focus in TB research has increasingly turned to these enzymes as new potential targets for chemotherapy. In this study, we isolated Mtb topoisomerase type IA (MtbTOPA), a subclass that only acts on single-stranded DNA (ssDNA), as our target of interest. At least one type IA topo activity must be present in every organism for resolving topological obstacles that require ssDNA cleavage, and fortuitously, Mtb happens to only encode one type IA, further validating MtbTOPA as a high-value drug target. We have cloned, expressed, and isolated highly purified recombinant MtbTOPA protein for enzymatic studies and work is currently underway to solve its crystal structure to aid in target-based approaches to drug development. In efforts to develop a high-throughput screen (HTS) for identification of small molecule inhibitors of MtbTOPA, we have collaborated with the Global Alliance for TB Drug Development (GATB) and leading structural genomics centers to gain access to large compound libraries as well as to accelerate the drug discovery process. We adopted a HTS method based on fluorescence resonance energy-transfer (FRET) and requisite MtbTOPA mutants were designed. It is our hope that the HTS will identify several hits that can be developed into novel TB drugs.

Leprosy, also known as Hansen’s disease, is a deteriorative skin condition caused by chronic infections of the bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Leprosy was once believed to be highly contagious, so patients with leprosy were quarantined and isolated to prevent further contamination within the population. In Korea, patients with leprosy were quarantined on Sorok Island where some patients received treatment for leprosy with the antibiotic, 4,4'-diaminodiphenylsulfone (DDS). Data collected on patients receiving DDS indicated that the average lifespans of the patients were longer than the average lifespans of the general Korean population suggesting that DDS may impact aging. Consistent with this idea, DDS has been reported to extend lifespan in the nematode Caenorhabditis elegans. We have confirmed these results in C. elegans, and the goal of this project is to identify the specific mechanisms and pathways through which DDS is able to extend lifespan. Our unpublished data support the model that one of the mechanisms underlying DDS’s effect on aging in C. elegans is activation of AMP- activated protein kinase (AMPK). We are currently working to understand how DDS activates AMPK, as well as the interaction between DDS and other known longevity pathways in C. elegans, such as dietary restriction and the hypoxic response. I am analyzing the mechanisms through which DDS affects aging by performing lifespan analysis on C. elegans and its mutants. By analyzing the effects of DDS on aging, we can get a better understanding of the mechanisms that control aging to eventually comprehend the pathways through which certain aging diseases, like Alzheimer's act.

Aging is a degenerative process characterized by progressive deterioration of cellular components resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in mammals. The specific mechanisms of longevity in C. elegans have not been completely identified, and many pathways are poorly understood. Dietary restriction (DR) is one promising pro-longevity intervention, though its mechanism is still poorly defined. DR, a reduction in caloric intake in absence of malnutrition, extends lifespan in C. elegans and in a wide range of other species including yeast, flies, mice, and primates. To investigate the genetic determinants of the response to DR, we are performing a genome-wide screen using the Vidal RNA-interference (RNAi) Library, which contains 11,511 unique RNAi clones. The C. elegans are fed E. coli that express dsDNA that, when ingested, form ribonucleic-protein complexes that bind to and facilitate cleavage of target mRNAs; effectively preventing expression of specific genes. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe extension or reduction in longevity in response to DR. We have screened over 8,143 RNAi clones; 71 of which reduce lifespan under DR conditions and 85 of which increase lifespan. We are currently following up on our data by verifying the effect of these RNAi clones on lifespan under both control and DR conditions. We hope to verify how significantly these genes are affected by DR and follow up on those specific pathways to further investigate the interaction between DR and other potential longevity pathways. These findings will provide insight into fundamental mechanisms of aging and may facilitate the development of therapies to delay the onset and progression of age-related diseases in people.