Thursday, May 28, 2015

From Herb Garden To Medicine Cabinet: Developing A New Drug for Malaria

We live on a lush planet filled with over 290,000 species of plants. Herbs are a particular type of plant that lack a wooden stem, and humans have often sampled them with hopes of finding a new food or flavoring. Sometimes ingestion of an herb produces unwanted effects, such as death. But other herbs have medicinal qualities, such as the alleviation of fever.

Dichroa febrifuga, a medicinal herb that has been historically used to treat fever, is named for its active ingredient, febrifugine.
Dichroa febrifuga is one of the most important herbs in traditional Chinese medicine, used for millennia to treat ailments such as malaria. Malaria is caused by a unicellular parasite called Plasmodium that is transmitted by mosquitoes, and a high fever is one of the trademark symptoms.

Malaria has a complex life cycle. After the parasites (sporozoites) are injected via mosquitoes, they travel to the liver (merozoites) and then infect red blood cells. In blood cells, they gobble up the hemoglobin as a nutrient source for replication and development into sexual stages (gametocytes) that can be taken up by another mosquito, thereby spreading the parasite to a new victim.
Malaria continues to be a devastating disease, killing up to 1 million people each year, most of whom are children under the age of five in sub-Saharan Africa. There is an urgent need for new treatments since the parasite has developed resistance to most of our anti-malaria drugs.

While effective against malaria, febrifugine is not tolerated well. What is needed is a better understanding of how febrifugine works:  how does it kill the malaria parasite? If the natural product’s mechanism of action against malaria could be identified, it would pave the way for the development of refined derivatives that are more specific against the parasite and less detrimental to patients. Alas, this is not an easy task. Over 2000 years in the making, scientists have now identified an enzyme in the parasite that is inhibited by febrifugine. That enzyme is called prolyl-tRNA synthetase.

Prolyl-tRNA synthetase is critical for the production of proteins in a cell, a process known as translation. As shown in the figure below, messenger RNA (mRNA), which serves as the “middle man” conveying the information in genes to build proteins, is read by molecular machines called ribosomes. Another type of RNA molecule called transfer RNA (tRNA) recognizes specific nucleotide sequences in the mRNA, bringing the corresponding amino acid to the ribosome so it can be added to a growing protein sequence.

The production of proteins in the cell. Proteins are composed of amino acids (the colored balls) that are connected together in a specific order, as directed by the gene coding for it. The chain of amino acids then typically folds into a three-dimensional shape so that the protein can do its job in the cell.
Aminoacyl-tRNA synthetase enzymes are needed to “charge” the tRNA; in other words, they attach the correct amino acid to the correct tRNA. When prolyl-tRNA synthetase is blocked by febrifugine, the amino acid proline does not get attached to tRNA. This leads to a buildup of “uncharged” tRNA, which is interpreted as a sign of starvation by the cell (or by the single-celled malaria parasite in this case). Proline is a common amino acid needed to build many proteins, and when prolyl-tRNA synthetase isn’t able to do its job, protein production grinds to a halt.

Even better, this enzyme is required in multiple stages of the parasite’s life cycle, knocking out both the liver and the blood forms. But as mentioned above, humans do not tolerate febrifugine very well, probably because we also have a version of prolyl-tRNA synthetase and perhaps other proteins that febrifugine poisons. Having identified this drug target is helping researchers develop derivatives of febrifugine, such as halofuginol, that act more strongly against the parasite’s prolyl-tRNA synthetase with less toxicity in humans.

Halofuginol is chemically similar to febrifugine (see above), having potent activity against malaria but less adverse effects on the host.
So how did scientists figure out that febrifugine targets prolyl-tRNA synthetase? There are several ways to identify the molecular mechanism of drug activity. In this case, the group cultured malaria in the presence of drug, forcing the parasites to evolve or die. Those that lived were less sensitive to febrifugine, meaning that they accrued a genetic change (one or more mutations in their DNA) that allowed them to persist despite the presence of the drug. This process is very analogous to the development of penicillin-resistant bacteria.

Parasites that were able to grow better in febrifugine had their genomes sequenced. Such a feat would have taken years and millions of dollars not long ago, but today it has become routine. The genome sequence of the febrifugine-resistant parasites contained a common mutation in the gene encoding prolyl-tRNA synthetase, which signaled that this enzyme plays a critical role in the drug’s action. Understanding how the parasite develops resistance also helps scientists design compounds that act on the target differently. As you may surmise, we are in a constant “arms race” with these insidious microbes, but this discovery is a step towards a victory for us.

Two independent parasite lines that were resistant to febrifugine, HFGR I and II, contained mutations in their prolyl-tRNA synthetase gene. In drug-sensitive parasites (Dd2), an amino acid called leucine (leu) is present at position 1444, but in the mutant parasites, a DNA change led to a different amino acid that conferred resistance to the drug.
Contributed by:  Bill Sullivan
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Herman JD, Pepper LR, Cortese JF, Estiu G, Galinsky K, Zuzarte-Luis V, Derbyshire ER, Ribacke U, Lukens AK, Santos SA, Patel V, Clish CB, Sullivan WJ Jr, Zhou H, Bopp SE, Schimmel P, Lindquist S, Clardy J, Mota MM, Keller TL, Whitman M, Wiest O, Wirth DF, & Mazitschek R (2015). The cytoplasmic prolyl-tRNA synthetase of the malaria parasite is a dual-stage target of febrifugine and its analogs. Science translational medicine, 7 (288) PMID: 25995223

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