Malaria is one of the most ancient and grievous human diseases and results in more than 1,000,000 deaths each year. The World Health Organization estimates that 300-500 million cases of malaria take place around the world. Most deaths occur in young children and pregnant women. Because malaria causes so many illnesses and death, it also has a devastating effect on many national economies. Since many countries with malaria are already among the poorer nations, the disease maintains a vicious cycle of sickness and poverty. Until recently, treatment of malaria with chloroquine was inexpensive and effective; however, resistance of Plasmodium falciparum (the causative agent) to this drug has spread from Asia to Africa making this drug ineffective. Chloroquine-resistance is also associated with reduced sensitivity to other antimalarial drugs such as quinine and amodiaquine. Successful treatment of this disease will therefore require development of new approaches. A collaborative project between our laboratories aims to develop new therapies by searching for drugs using a simple high throughput assay that employs derivatives of substrate NTPs and mitochondrial RNA polymerase, a key enzyme involved in mitochondrial gene expression of the malaria parasite. Development of this assay would allow efficient screening of hundreds of thousands of chemical compounds to find potential antimalarial drugs.
The first step in gene expression, transcription of the information in the DNA genome into messenger RNA, is carried out by RNA polymerases (RNAPs). These enzymes therefore play a key role in all organisms, and have become attractive targets in the development of antibiotics. For example, the front-line anti-tuberculosis drugs rifampin and rifampicin effectively inhibit bacterial RNAPs. A number of other potent inhibitors that affect different stages of transcription or target different regions of the bacterial RNAP have been reported, such as streptolydigin, microcin, and myxopyronin. These proteins are thus attractive therapeutic targets in the search for new antibiotics.
In addition to the large multi-subunit nuclear RNAPs, eukaryotic cells also have a smaller mitochondrial RNAP (mitoRNAP) that is responsible for transcription of the genome of the organelle. While differences between human nuclear RNAPs and nuclear RNAPs of single cell eukaryotic parasites can, in principle, be exploited to find specific inhibitors, the high structural and relatively high sequence homology between these enzymes will likely make this task difficult. In contrast, mitoRNAP of single cell eukaryotes shares very limited homology with human mitoRNA polymerase, and likely operates using a quite distinct set of transcription factors and promoters. We propose that there should be compounds that specifically inhibit Plasmodium mitoRNAP (or other components of mitochondrial transcription) but have little or no effect on transcription by human mitoRNAP. Because the viability of the parasite depends on mitochondria function, it is reasonable to expect that specific prevention of mitochondrial gene expression and replication will result in death of the parasite.
All mitoRNAPs appear to have arisen from an ancient bacterial endosymbiont, and are related to a well characterized RNAP encoded by a bacterial virus (phage T7) that is structurally distinct and shares no sequence homology with large multi-subunit nuclear RNAPs. It is therefore expected that such compounds will have minimal toxicity associated with their effects on nuclear transcription.
The genome of Plasmodium mitochondria is one of the smallest genomes and encodes mRNAs for only three proteins (Figure 1). Transcription of this genome is carried out by nuclear-encoded mitoRNAP which is assisted by at least one transcription factor. A strategy to develop a fluorescent high throughput screening assay to examine large libraries of chemical compounds for specific inhibitory activity will be based on the ability of recombinant Plasmodium mitoRNAP, obtained in Dr. Temiakov’s laboratory to carry out promoter-independent RNA synthesis and utilize fluorogenic substrate analogs. All known RNAPs can synthesize RNA on templates containing runs of poly (A)-poly(dT) sequences eliminating the need for specific transcription factors and regulatory DNA regions (promoters).
A fluorescence-based assay for RNAPs previously developed in Dr. Mustaev’s laboratory utilizes nucleoside triphosphate substrates with substitutions at the -phosphate position that introduce a fluorescent 4-methylumbelliferone moiety (Figure 2). Incorporation of the nucleoside monophosphate residue of these compounds into RNA is accompanied by the release of the pyrophosphate derivative of 4-methylumbelliferone. Subsequent treatment with alkaline phosphatase releases the umbelliferyl anion, which is highly fluorescent and can be easily detected. The unincorporated portion of the umbelliferone NTP derivative is resistant to phosphatase. It is not fluorescent, and therefore does not contribute to the signal. It is expected that the intensity of the final fluorescent signal would reflect RNAP activity. We have chosen 4-methylumbelliferone because it has been commonly used in enzymatic assays due to high fluorescence and because of its relatively small size, which would allow the NTP derivative to be utilized as an RNAP substrate. Originally this assay was developed for bacterial RNA polymerases. In the present work we demonstrated applicability of the assay for mitochondrial RNA polymerase. This opens the possibility to use the assay for a search of the inhibitors of mitoRNAP using high throughput screening. Identification of inhibitors of this key enzyme would enable targeted drug design and provide new drugs for efficient
Dmitry Temiakov is an assistant professor in the Department of Molecular Biology at UMDNJ-School of Osteopathic Medicine. He received his MS degree in biotechnology in 1993 from Mendeleev University, Moscow, Russia, and his PhD in molecular biology in 1996 from the Institute of Genetics, Moscow, Russia. He pursued his post-doctoral training at the Downstate Medical Center in Brooklyn, NY. Dr. Temiakov is currently involved in studies of molecular mechanisms of transcription regulation in human mitochondria.
Arkady Mustaev is an assistant professor in the Department of Microbiology and Molecular Genetics at UMDNJ- New Jersey Medical School. He received his PhD in 1987 from Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences. His post-doctoral training was done at the Limnological Institute, Irkutsk, Russia, as well as at Columbia University and the Public Health Research Institute, NY. Currently Dr. Mustaev is involved in drug discovery projects, development of highly sensitive fluorescent probes for biological applications and in studies of the molecular mechanisms of prokaryotic transcription.