The Post-Chloroquine Era

Figure 1. Chinchona bark

containing quinine (9).

by FC

Nothing in this world seems as interminable as the race of humans versus pathogens. The moment we celebrate a success against an organism that plagues our existence we are served another. Just when we find a weapon to combat the pathogen, it changes its attack. This pattern is no exception for Plasmodium falciparum. Earning the title as one of the most deadly infectious parasites, its widespread occurrence is a health and economic issue (4). For some time the advent of chloroquine allowed us a triumph, but today a grimmer picture is portrayed. Not only has chloroquine use become obsolete, the alternatives are limited by cost and supply (4,7). Unless serious international investment and multinational collaboration takes place, the rising health and economic burden of malaria will continue unabated (4).

When chloroquine came onto the scene in 1934, it addressed the demand for a quinine substitute (7,8). Found in Chinchona tree bark, quinine was used for an estimated 278 years before reports of P. falciparum resistance began to surface around 1910 (5,8). Efforts to synthesize a new antimalarial surged in the early 20^th century, producing the worlds’ most widely used antimalarial. However, this advancement did not occur without setbacks. When chloroquine was first synthesized it was dismissed on the assumption that it was too toxic. Its resurrection was triggered only when the Japanese cut off the world supply of quinine in World War II, and by 1946 studies by American researchers observed its utility as a powerful antimalarial (3).

Figure 3. World map highlighting regions with

 a chloroquine failure rate of 10% or more (10). 

Contrary to its popularity, chloroquine did not enjoy the same lengthy period of effectiveness as quinine did (7). It was 12 years after its widespread use when reports of chloroquine resistance began to appear (8). As shown in Figure 3, chloroquine resistance currently occurs worldwide, rendering it an ineffective treatment (3). However, newer drugs have experienced even less longevity: mefloquine resistance occurred five years after its distribution, and atovaquone barely saw a year pass by (8).

What gave chloroquine such a relatively long run is its mechanism of action. Newer compounds target enzymes, contributing to their rapid ineffectiveness. Chloroquine’s mechanism of action is unique because it is non-enzymatic (5). Plasmodium falciparum contains a digestive food vacuole that breaks down hemoglobin as it grows in our red blood cells. The breakdown product from hemoglobin digestion is hematin and highly toxic to P. falciparum. To cope with the toxicity P. falciparum clumps hematin into harmless crystals and stores them within the vacuole. Chloroquine disrupts the polymerization process by binding to hematin and maintaining it in its toxic form, killing the parasite by accumulation (5). This metabolic pathway appears to be rather complex because P. falciparum has to undergo several mutations before resistance is conferred. This complexity appears to be missing in regard to drugs like pyrimethamine, where resistance is easily acquired via a single mutation in an enzyme (7).

With drug resistance appearing in newer drugs at a rate faster than the time invested in researching and developing these agents, options seem limited. There is a need for new drug targets and new therapies, and recently significant progress has been made to meet this need.

Drug development can be approached using several tactics.  One method is to develop analogs of existing agents. The benefit is that the relative effectiveness and mechanism is already known but the slight different can circumvent resistance problems.   It was by this method that chloroquine was synthesized from quinine and subsequently several other quinine-related compounds such as tafenoquine and the 4-aminoquinolines (5). As an example, a group of researchers recently focused on quinolones, a compound unique in its ability to perform inhibitory function in two discrete cell pathways. While inhibiting bc₁, a component of cellular respiration, appropriately structured quinolones can also stack together and chelate heme, depriving P. falciparum of both a method of creating energy and obtaining nutrients. From a base template they developed twenty novel quinolone derivatives that were tested for effectiveness against P. falciparum and found a quinolone ester derivative that was highly effective (1).

Finding compounds in the natural environment is another option. While compounds isolated from natural sources may make it easier to introduce a treatment to natives, the cost is relatively high compared to other methods (5). One drug found in this manner is artemisinin. In the 1980s drug-resistant malaria affecting both sides of the Vietnam War spurred both China and the United States to develop new effective drugs. Chinese scientists discovered the compound artemisinin in sweet wormwood, now known in the form of derivatives such as artemether, arteether, and artesunate (4). Like chloroquine, this highly effective drug also targets P. falciparum’s food vacuole and stimulates free-radial formation that inflicts damage to the cell. To this date, P. falciparum remains sensitive to artemisinin, making it an ideal replacement for chloroquine use (8).

Until recently a significant amount of research has focused on the interaction between P. falciparum and humans and as a result all drugs and agents on the market so far have been developed based on this relationship. However, the complex lifecycle of P. falciparum makes it possible to study mosquitos as potential drug targets as well. This is particularly important because mature gametocytes can persist in a patient’s blood for weeks after alleviation of symptoms, thus transmission can still occur if a mosquito takes its blood meal in that time period. Earlier this November a research group reported a class of compounds that blocks oocyte development in the midgut of the mosquito (2). This halts P. falciparum transmission to a new host because midgut oocyte development is the precursor of sporozoites (7). These sporozoites travel to the mosquito salivary glands and infect a person bitten by that mosquito. Table 4, taken from this paper, shows the effect of several drugs on oocyte counts in the mosquito midgut. They were able to identify cyproheptadine and protryptyline as highly effective compounds against midgut oocyte development and these compounds were also considered safe for humans and animals. If this research is confirmed and an effective method of distribution of the compound is elucidated, it will have significant implications in new methods of P. falciparum control.

Even with all these advances P. falciparum’s ancestral relatedness to us makes it difficult to identify drug targets that are will not also negatively affect our own cells. In order to accomplish this an improved understanding of the physiology of P. falciparum is required. Even though the genome sequence is readily available biochemical and genetic manipulations are still a limiting factor in ongoing research (5). This adds to the complexity of developing new drugs and drug targets.

            The development of antimalarial agents gets even more complicated from an economical standpoint. Ironically the populations who need an effective antimalarial treatment the most are the ones that have next to no funds to cough up for it. Lack of private investment in current antimalarial drug development and distribution can be blamed on this irony. Companies would rather pursue treatments for what I like to call “first world problems” which guarantee a consumer base that is able to afford the costs of these treatments (4). Such is not the case for a vast majority of malaria sufferers worldwide.

Simple economics is the predominant reason why chloroquine was such an effective treatment. At $0.10 an adult dose and orally ingested, chloroquine was both affordable and easily administered (4). Currently the danger with chloroquine is that although ineffective it is still being distributed in resistant regions of Africa instead of newer and more effective therapies because of the cost. At its cheapest, even artemisinins are at least ten times as expensive for consumers as chloroquine. It is estimated that in order to make artemisinins affordable subsidies of $300 to $500 million a year are required. If that sounds like a lot of money, it pales in comparison to the economic burden malaria has in Africa. Lost productivity and investment revenues due to P. falciparum-related illness and death amounts to an estimated $3-12 billion a year in the continental gross domestic product. (4).

 Although it is present, benevolent charity contributions can only go so far. In order to create incentive for pharmaceuticals and other agencies to develop antimalarials there has to be a market worth investing in. A report issued by the Institute of Medicine Committee on Economics of Antimalarial Drugs suggests the $300 to $500 million subsidies mentioned before so artemisinin becomes affordable for consumers. An additional $10 to $30 million is also recommended for short-term production stimulation. The idea is that an international organization purchases the treatment from the producing companies for the competitive market price, and sells the drug to countries’ governments at the same price chloroquine is sold, thereby absorbing the cost difference. In return, countries ensure that the treatment is properly distributed to replace chloroquine in even the most impoverished regions. If these suggestions are put into action, it will increase access to effective antimalarials in impoverished areas. This will allow us to regain control of P. falciparum and alleviate the economical and social burden malaria has in these endemic regions (4).

References:

1.       Cowley, R., Leung, S., Fisher, N., Al-Helal, M., Berry, N. G., Lawrenson, A. S., ... & Paul, M. O. (2012). The development of quinolone esters as novel antimalarial agents targeting the Plasmodium falciparum bc1 protein complex. MedChemComm, 3(1), 39-44.

2.       Eastman, R. T., Pattaradilokrat, S., Raj, D. K., Dixit, S., Deng, B., Miura, K., ... & Su, X. Z. (2012). A Class of Tricyclic Compounds Blocking Malaria Oocyst Development and Transmission. Antimicrobial Agents and Chemotherapy.

3.       Meshnick, S. R., & Dobson, M. J. (2001). The history of antimalarial drugs. Antimalarial chemotherapy, 15-25.

4.       Panosian, C. B. (2005). Economic access to effective drugs for falciparum malaria. Clinical infectious diseases, 40(5), 713-717.

5.       Rosenthal, P. J. (2003). Antimalarial drug discovery: old and new approaches. Journal of experimental biology, 206(21), 3735-3744.

6.       Vernick, K. D., & Waters, A. P. (2004). Genomics and malaria control. New England Journal of Medicine, 351(18), 1901-1904.

7.       Wellems, T. E., & Plowe, C. V. (2001). Chloroquine-resistant malaria. Journal of Infectious Diseases, 184(6), 770-776.

8.       Wongsrichanalai, C., Pickard, A. L., Wernsdorfer, W. H., & Meshnick, S. R. (2002). Epidemiology of drug-resistant malaria. The Lancet infectious diseases, 2(4), 209-218.

9.       [Untitled photograph of chinchona bark] Retrieved November 14, 2012, from: http://upload.wikimedia.org/wikipedia/commons/thumb/0/08/Cinchona_officinalis_001.JPG/220px-Cinchona_officinalis_001.JPG

10.    [Untitled world map of chloroquine resistance] Retrieved November 14, 2012, from: http://helid.digicollection.org/en/d/Js13418e/14.6.html

Friend or Foe? The Paramecium-Algae story

by SC

A relationship has been found in nature between Paramecium bursaria, a ciliate protist, and chlorella, a green algae, who have been known to help one another in certain situations (image shown below, the larger cell being P. bursaria and the green organisms inside the cell are chlorella). This is an endosymbiotic relationship in which Chlorella resides inside P. bursaria for food in exchange chlorella gives P. bursaria the by-products of photosynthesis. Symbiosis has been known to hold a huge role in biology; for example, mitochondria in eukaryotic cells are theorized to have once been free living bacteria that now serve as the eukaryotic energy producer. In this blog post we will learn how prey can become a beneficial part of the cell and what happens when chlorella stops benefiting P. bursaria in its endosymbiotic relationship.

Paramecium bursaria

P. bursaria provides nitrogen, carbon dioxide, and protection from viruses that target chlorella and in return chlorella provides a food source to the ciliate by producing sugars as a by-product from photosynthesis. It has been studied that not only does P. bursaria protect chlorella from viruses that want to infect it, but chlorella can also return the favor by protecting P.bursaria in a different way. The results from a study show that chlorella can protect P. bursaria from UV radiation from the sun [1]. UV radiation can cause lethal mutations in a cell’s genome, which can inhibit its ability to perform photosynthesis and inhibit growth. P. bursaria strategically places the chlorella near the edges of the cell and around its macronucleus, a large nucleus found in ciliates, to prevent the UV radiation from getting to the DNA and damaging the host’s genome. Evidence from the summerer paper et al. paper supported this conclusion by exposing symbiotic P. bursaria, P. bursaria with reduced chlorella, and P. bursaria with no chlorella to UV radiation. The results showed that when exposed to UV radiation both the symbiotic P. bursaria and P. bursaria with reduced chlorella showed growth in the presence of UV radiation while the P. bursaria with no chlorella showed a significant decrease in the number of cells that were still alive after the UV exposure (As seen in the figure below). Both of these organisms are capable of living independently of each other, they have been used in experiments to recreate the genesis of their symbiotic relationship.

Figure

Researchers have used two independent strains of chlorella and P. bursaria and found that they were able to create the symbiotic relationship found in nature in 30 minutes [2]. For clarification, Chlorella is found in the parialgal vacuole of P. bursaria, which is derived from a P. bursaria digestive vacuole. When Chlorella is ingested it enters a digestive vacuole and must prevent the fusion of the lysosomes to the vacuole. If they were to fuse lysosomal enzymes would cause the degradation of chlorella. The chlorella that succeeds in preventing fusion starts its new symbiotic relationship with P. bursaria . The first step is the phagocytosis of chlorella into the P. bursaria cell, which is placed in an acidic digestive vacuole that has the ability to bind to lysosomes for further digestion of the chlorella. At first, P. bursaria is simply trying to acquire a meal, but occasionally the algae is able to bud from the digestive vacuole before the lysosome binds to the vacuole. It would appear at this point that the chlorella has control of where this vacuole goes since it migrates right underneath the host cell membrane. Tests by Kodama et al. show that time of budding to the time it reaches the edge of the cell membrane the vacuole differentiates and is controlled by the chlorella. At this point the vacuole can no longer be combined with a lysosome and P. bursaria and chlorella start their new symbiotic life together. If chlorella stops doing its job it will be degraded and it will be digested by P. bursaria.

Another paper by Kodama et al. suggests that when the protein synthesis of chlorella stops working P. bursaria will break off its symbiotic relationship and digests chlorella. This reaction was studied by introducing a solution of cycloheximide to selective media that affects protein synthesis of chlorella but does not affect P. bursaria enough to be considered important. Nine hours after cycloheximide is introduced to the media, P. bursaria is able to fuse lysosomes to the algal vacuole and digest the chlorella. The results gathered show that when the proteins in chlorella stop working it can no longer maintain its chloroplast and nucleus structure; consequently these organelles are degraded and then digested by P. bursaria.

With this information, it can be said that P. bursaria has a selfish relationship with chlorella. When the two organisms first meet P. bursaria eats chlorella and places it in a situation where if chlorella survives it will live the rest of its life inside P. bursaria. If unsuccessful, P.bursaria will digest chlorella for food. This is a win-win situation for P. bursaria because either way it will acquire a meal; however, it is more beneficial to keep chlorella alive since it provides a food source to P. bursaria as long as the paramecium provides food for the algae. P. bursaria also benefits from the UV protection provided by chlorella. At the end of chlorella’s life the organelles will degrade and be digested by P. bursaria for food. For one final point it should be noted that during chlorella’s life it will reproduce so this abusive cycle will be passed down through its progeny, but since it is a cell it doesn’t think like this all it knows is that it is getting food from its captor... Can anyone say Stockholm Syndrome?

References:

1.     Summerer, Monika, et al. "Symbiotic Ciliates Receive Protection Against UV Damage from their Algae: A Test with Paramecium bursaria and Chlorella." Protist 160.2 (2009): 233-243.

2.     Kodama, Yuuki, and Masahiro Fujishima. "Timing of Perialgal Vacuole Membrane Differentiation from Digestive Vacuole Membrane in Infection of Symbiotic Algae Chlorella vulgaris of the Ciliate Paramecium bursaria." Protist 160.1 (2009): 65-74.

3.     Kodama, Yuuki, Isao Inouye, and Masahiro Fujishima. "Symbiotic Chlorella vulgaris of the Ciliate Paramecium bursaria Plays an Important Role in Maintaining Perialgal Vacuole Membrane Functions." Protist 162.2 (2011): 288-303.