Malaria: The Disease and Parasite Plasmodium falciparum

Part 1: Malaria: The Disease and Parasites

00:00:00.00 Hi, my name is Joseph Derisi and I am an associate professor in the department of biochemistry,
00:00:06.26 biophysics, and a Howard Hughes Investigator at the University of California, San Francisco.
00:00:11.18 The subject of my lecture today is malaria.
00:00:14.13 And it is my goal to give you a background, and general overview of the disease,
00:00:18.23 and the parasites that cause this deadly affliction.
00:00:21.05 So first, let's talk about the disease.
00:00:24.08 The disease malaria is a devastating condition, it is caused by one of four parasitic protozoa.
00:00:31.15 It's characterized by a periodic fever,
00:00:34.24 and/or a febrile incident. This is usually followed by chills, sweats, aches, and other devastating conditions.
00:00:43.07 It is also characterized by enlargement of the spleen, and in the worst form,
00:00:47.00 so called cerebral malaria, can result in seizures, comma, severe anemia,
00:00:51.27 and ultimately organ failure and death.
00:00:54.08 It is true that children are the most affected by this disease and most likely to die.
00:01:00.05 The toll of human malaria is incredible.
00:01:03.15 There is somewhere between 1 and 2 million deaths due to this disease per year.
00:01:08.12 And that is in addition to 4 million to 9 million disease incidents per year.
00:01:14.00 So let me put that in perspective for you.
00:01:16.03 One million deaths per year equates to someone dying of this disease
00:01:20.00 every 30 seconds, of every minute, every day of the year.
00:01:23.26 And so, it can not be understated how incredibly this disease is on humanity.
00:01:31.29
00:01:33.26 So, why is malaria such a problem?
00:01:38.07 Why are we still dealing with this today,
00:01:40.05 when the cause of this disease has been known for over 100 years?
00:01:43.29 Well, it is simple. Today, malaria enjoys world-wide drug resistance.
00:01:51.01 That is the limited medications that we have to treat the disease,
00:01:54.29 are useless in many parts of the world.
00:01:57.01 And I'll have more to say about this in a little bit.
00:01:59.08 There is currently no vaccine for this disease with any operation impact.
00:02:03.17 And, to acquire some limited immunity to the disease,
00:02:07.19 requires that people be repeatedly infected, on a very frequent basis.
00:02:13.28 Therefore, to have any immunity to the disease requires continual pressure by the parasite.
00:02:21.14 No vaccine that has been created today achieves the same level of protection as natural infection.
00:02:28.15 And that is a problem.
00:02:30.16 So, this is where in the world the risk of transmission of malaria is the most severe.
00:02:39.12 Colored in dark green, are those areas of the most transmission of the disease.
00:02:44.08 And as you can see, here in sub-Saharan Africa, the problem is the worst.
00:02:49.02 Now, this is not to say that the problem isn't bad in South America, Central America, and South-east Asia,
00:02:54.18 it is. But by far and away, the largest burden to malaria is sub-Sahara Africa.
00:02:59.27 And the disease malaria is transmitted by the Anopheles mosquito.
00:03:04.29 There are over 60 species of the Anopheles mosquito,
00:03:08.08 that are capable of transmitting the disease.
00:03:10.10 This is why it is so difficult to control.
00:03:13.22 You might think that one way to control the disease
00:03:16.25 would simply be to eradicate the mosquitoes that carry it.
00:03:19.17 This is what has worked in many first world countries that have the organization,
00:03:23.18 the money, and the governmental structure to support such an effort.
00:03:27.00 And in fact, during the 1950s through the 1970s,
00:03:31.19 there was a world-wide global eradication effort led by WHO,
00:03:36.00 the World Health Organization.
00:03:37.10 And here is a post card from the United States
00:03:39.26 commemorating the initiation of the world-wide eradication effort.
00:03:43.25 But, since I'm giving you this lecture,
00:03:46.00 you know that this eradication effort was doomed to failure.
00:03:49.02 Why did it fail? It failed for a variety of different reasons that are illustrated here.
00:03:52.23 First of all, as I mentioned before,
00:03:54.23 there is drug resistance to the medications we use to treat the sick.
00:03:58.04 Furthermore, a lot of the eradication effort bypassed Africa
00:04:02.08 where the worst problems with malaria were occurring.
00:04:04.14 Furthermore, there was a lack of sustainable funding for this effort.
00:04:09.01 And, along with that, a lack of sustainable community participation in the eradication effort.
00:04:14.25 This was compounded and complicated by the fact
00:04:18.11 that there was massive population movements throughout the world,
00:04:21.07 in areas endemic with malaria,
00:04:22.28 and war, which never helps anything.
00:04:26.05 Furthermore, insects became resistant to many of the insecticides used
00:04:31.02 to try to eradicate the vectors themselves.
00:04:33.16 So what about the parasites themselves?
00:04:36.03 What is it we are trying to treat?
00:04:38.28 Well, as I mentioned before, the disease malaria is caused by any one of four protozoa parasites.
00:04:46.00 And their names are right here.
00:04:47.22 The worst, and by far the most deadly one, is Plasmodium falciparum.
00:04:52.02 Followed by Plasmodium vivax, Plasmodium malariae, Plasmodium ovale.
00:04:57.24 Plasmodium falciparum, as I mentioned,
00:05:00.05 is the most deadly of the four, and has the largest distribution worldwide.
00:05:05.20 Now, if you were to take some blood from an infected individual, smear it on a glass slide,
00:05:11.13 and stain with dye that binds DNA, you would see something like this.
00:05:17.06 Now, as you may know, red blood cells, or erythrocytes,
00:05:20.07 do not contain nuclei, and thus do not contain DNA.
00:05:23.21 But the parasites do.
00:05:25.07 So, if we did stain with a dye that bound DNA,
00:05:28.18 you would see healthy red blood cells, such as this one and this one.
00:05:32.04 But you would also see red blood cells with dark objects on the inside,
00:05:36.08 such as here and here.
00:05:38.00 These are Plasmodial parasites. In fact this is Plasmodium falciparum.
00:05:42.10 And this is the most frequent way an infection by Plasmodium is diagnosed.
00:05:47.19 The so called blood smear.
00:05:49.04
00:05:50.19 So let us talk more about the life cycle of these parasites,
00:05:54.26 and how they go about creating this horrible disease.
00:05:57.13 The life cycle is shown in cartoon form here.
00:06:01.19 Now, this is oversimplified for the purpose of this lecture,
00:06:04.25 but I just want to hit on the main points of the developmental cycle.
00:06:08.22 So it begins with the bite of the mosquito,
00:06:11.11 as I mentioned the Anopheles mosquito carries the Plasmodium parasites.
00:06:14.23 The Anopheles mosquito then releases sporozoites into the blood stream.
00:06:20.00 These sporozoites travel through the blood stream to the liver,
00:06:23.19 where they then invade hepatocytes.
00:06:25.19 It is here that the parasites replicate,
00:06:28.13 and develop over the course of a little more than a week.
00:06:31.16 After this period of replication,
00:06:34.04 the so called merozoites burst forth from the hepatocyte,
00:06:38.03 and begin a continual cycle of infecting red blood cells.
00:06:42.18 The so-called asexual stage of the parasite.
00:06:45.11 It is here that the parasites will invade red blood cells,
00:06:50.01 and consume the hemoglobin inside as a food source.
00:06:52.23 This is where all the clinical manifestations of the disease occur,
00:06:56.24 and this is where most drugs attempt to eradicate the parasite.
00:07:01.01 Now, with some infrequent rate, by a mechanism that is frankly poorly understood,
00:07:07.02 a small number of parasites will develop into a sexual form called gametocytes.
00:07:11.10 It is these gametocytes that are then picked up by a new anopheles mosquito
00:07:16.07 when taking up a blood meal,
00:07:18.02 and the entire cycle starts again.
00:07:20.04 And so, if one can treat the disease either here,
00:07:25.22 or block transmission here,
00:07:28.06 or block passing on the disease to new mosquitoes here,
00:07:31.21 one can effectively control this disease.
00:07:34.13 Now, let's take a little more close look at each stage here.
00:07:39.03 So I'd like to show you a movie first.
00:07:40.21 So in this movie by Vandenberg and colleagues,
00:07:44.13 which you will see is the proboscis of a mosquito that is the needle-like, tube-like structure
00:07:49.28 that the mosquito uses to suck the blood of its victim,
00:07:53.01 piercing the skin of a mouse,
00:07:55.20 and what you will observe are the infective form of the malaria parasites,
00:08:01.00 the sporozoites, being released from the tip of the proboscis into the saliva.
00:08:05.17 So let me start the movie and guide you through it.
00:08:08.21 So first, what you will see,
00:08:10.20 is the proboscis moving around trying to find a good blood supply.
00:08:14.28 Then, a second mosquito comes on the scene,
00:08:17.12 and you will see the proboscis over here.
00:08:19.16 See these small white particles, that seem to be moving around?
00:08:23.28 These are the sporozoites, and they exhibit what is called gliding motility.
00:08:28.05 As they try to find the blood stream,
00:08:30.24 and ultimately seek out the liver and invade hepatocytes.
00:08:34.06 We will let that movie run for a second so you can see the sporozoites circle around.
00:08:40.07 And then finally, the proboscis of this mosquito will withdraw.
00:08:44.15 O.K. So, back to the life cycle.
00:08:48.12 You have seen sporozoites enter the blood stream,
00:08:50.26 from here they go to the liver, replicate,
00:08:53.04 and then burst forth in a multitude of numbers to infect erythrocytes,
00:08:57.21 in essentially a perpetual asexual cycle.
00:09:02.12 And it is here, that again, all the clinical manifestations of the disease occur.
00:09:06.16 So, let's focus on that blood stage, asexual cycle of the parasite.
00:09:12.03 This is it shown in more detail.
00:09:15.02 As I mentioned before, this is the form that bursts forth from the liver, the so called merozoites.
00:09:20.09 It is these small forms of the parasite that will bind to a new erythrocyte and invade it.
00:09:25.23 Now, after invasion the parasite is called a ring stage,
00:09:30.00 and that is because it resembles a small diamond ring on the inside of the erythrocyte.
00:09:33.29 Then, the main metabolic phase of the parasite follows,
00:09:37.20 this is the so-called trophozoite phase,
00:09:39.27 and after this, the parasite undergoes multiple rounds of replication,
00:09:44.06 to ultimately form what is called a schizont.
00:09:47.09 A schizont will then mature and burst again,
00:09:50.27 and merozoites are released,
00:09:52.11 and this entire cycle repeats itself essentially every 48 hours in Plasmodium falciparum.
00:09:58.16 So, let's focus a little bit more on this stage, and this stage with yet another movie.
00:10:05.01 In this movie, by Glushakova and colleagues,
00:10:09.15 what you will see is an infected erythrocyte shown here,
00:10:13.06 burst spilling forth merozoites.
00:10:16.05 This erythrocyte that is uninfected over here,
00:10:19.08 will become infected by a new merozoite that attaches to it,
00:10:23.13 and then enters through the membrane.
00:10:25.21 So, as I start this movie, you will see this dark spot in the middle,
00:10:30.04 this a crystal of so-called hemozoin, this is the by product of hemoglobin digestion,
00:10:35.12 that has been collected in the food vacuole of the parasite.
00:10:38.24 So let's start the movie.
00:10:40.12
00:10:42.22 The erythrocyte bursts, and now you can se the merozoites.
00:10:48.09 Now look closely here.
00:10:49.15 There goes a merozoite into the cell. Did you see that?
00:10:53.01 It actually happens quite fast.
00:10:54.22 Now, the real time of this movie is shown in the upper left corner.
00:10:57.29 Let's see it again.
00:10:59.15 The erythrocyte bursts, and watch here.
00:11:01.17 There it is.
00:11:03.29 The merozoite enters the blood cell.
00:11:06.08 So in a matter of about less than 120 seconds,
00:11:09.19 cells burst, merozoites find new homes,
00:11:13.04 invade, and yet the process starts again.
00:11:16.00 And as you can see, if one parasite enters a cell, and many more come out,
00:11:21.16 upwards from anywhere 12 to 24,
00:11:24.25 you can imagine that this exponential increase in parasites is devastating to the human host.
00:11:30.17 And you can also imagine how complications like severe anemia can occur.
00:11:35.05
00:11:36.25 OK. Let's go back to the life cycle.
00:11:39.16 As I mentioned before,
00:11:40.28 the asexual erythrocytic cell cycle, of the Plasmodium parasites,
00:11:45.10 is where all the clinical manifestations of the disease occur.
00:11:48.20 So, if we are seeking to control malaria,
00:11:51.23 at the level of treating patients, people sick with the disease,
00:11:55.27 this is the stage which drugs must be designed to work against, to have efficacy.
00:12:01.27 So let me give you a quick review of the historical nature of drugs used against malaria.
00:12:08.27 Now, I'm not going to cover all of the drugs,
00:12:10.23 but some of the milestone drugs in malaria development.
00:12:14.04 So, we can begin in the 1600s with quinine.
00:12:18.07 This is the most famous of all malaria drugs,
00:12:20.23 and it comes from the bark of a particular tree found in South America.
00:12:24.28 It was brought to the new world by the Jesuits,
00:12:27.04 and for the last 400 years, it has been the mainstay of malaria treatment.
00:12:32.09 Now that being said, there are instances of quinine resistance throughout the world.
00:12:37.15 Now, during the 1940s, actually, during World War 2,
00:12:42.20 there became a need for additional malaria drugs,
00:12:46.07 because quinine supplies,
00:12:48.07 were not available to allied forces because they came under the imperial Japanese control
00:12:52.19 Chloroquine, was a German drug, that was rediscovered by Walter Reed, of the US Army,
00:12:58.21 during an effort to find new anti-malarial medications.
00:13:02.25 I should mention that many of the wars throughout history
00:13:06.27 have been shaped by malaria, and the disease that it causes.
00:13:10.15 And so there is great interest to the US Army, who puts troops in harms way,
00:13:15.07 in places where malarial transmission is endemic, to find new treatments.
00:13:20.14 Chloroquine is one of the wonder drugs of its time.
00:13:23.25 It was very cheap to make, very bio-available, and extremely potent against Plasmodium falciparum.
00:13:31.00 This was the drug of choice for a long time.
00:13:34.08 But unfortunately, due to misuse, and frankly, overuse throughout the world,
00:13:38.19 it became useless in South America and Southeast Asia,
00:13:42.28 and then resistance spread worldwide.
00:13:44.21 Which is why, in the 1970s during the Vietnam war, Walter Reed, the US Army, once again embarked on large screening programs.
00:13:52.23 Out of which came the new drug mefloquine.
00:13:55.21 Also known as lariam.
00:13:57.20 Mefloquine has been the mainstay for the use against drug-resistant parasites for some time.
00:14:03.21 Although now, there are parasites, again spread worldwide,
00:14:07.13 that have shown mefloquine resistance.
00:14:10.03 This is also a drug that is not as cheap or easy to use as chloroquine,
00:14:13.27 and so it is not ideal.
00:14:15.18 Out of the very same screening efforts that brought us mefloquine,
00:14:18.27 also came halofantrine.
00:14:20.26 This is a drug that was being investigated during the 1980s for use,
00:14:25.21 it was shown to bypass resistance to chloroquine and mefloquine resistant parasites.
00:14:30.22 Yet, halofantrine also has its issues. Primarily, cardio toxicity in certain individuals.
00:14:36.18 And so, it cannot be considered as a drug for ultimate widespread use.
00:14:42.01 Now, we are here in the 1990s, and the drug of choice for researchers to investigate is artemisinin.
00:14:49.14 Artemisinin is a compound that was originally discovered in a Chinese herbal remedy,
00:14:55.18 for malaria,
00:14:56.16 as part of a screening program on the part of the Chinese army,
00:14:59.21 and was later validated by Walter Reed in the US Army and others.
00:15:04.02 Now, as you can see from this timeline,
00:15:07.25 over the last 400 years, and these are not all the malaria drugs,
00:15:10.28 but over the last 400 years, there has been very few compounds developed for malaria.
00:15:16.26 And that is because malaria is endemic in the poorest parts of the world.
00:15:20.11 And there is not a very large profit incentive for major pharmaceuticals to develop.
00:15:25.14 This is beginning to change,
00:15:27.06 now with the advent of greater philanthropy for malaria research and therapeutic development.
00:15:33.14 Yet, I want to impress upon you,
00:15:35.11 that very few drugs are developed,
00:15:37.26 every year or even every decade
00:15:39.29 for anti-malarial use. And, I should also mention that the days of mono-therapy are over.
00:15:47.23 It is not possible for us to rely on a single drug,
00:15:50.27 to help eradicate this scourge. And from the lessons of HIV and other infectious disease,
00:15:56.24 we have learned that to bypass resistance,
00:15:59.03 or to lower the probability that resistance will occur in new parasites,
00:16:03.17 we must used drugs in combinations.
00:16:06.02 And so, artemesinin is being used in combination.
00:16:10.00 This is a formulation called Coartem,
00:16:12.17 which is a mixture of artemesinin and lumifantrane, another drug,
00:16:16.19 That can be used in combination
00:16:19.03 to help lower the probability that parasites will become resistant to this drug.
00:16:23.19 Now, these compounds are not easy to make and not cheap to make,
00:16:28.07 which is why there is limited availability of Coartem
00:16:31.27 throughout the developing world right now.
00:16:34.05 But if we are ever to hope to control malaria,
00:16:37.03 we need to treat people that are sick, and we need combination therapies.
00:16:41.08 So, a large emphasis of research in the malaria community,
00:16:45.00 and in my lab, is to help try to develop new, cheap,
00:16:49.04 bio-available, non-toxic anti-malarial therapeutics.
00:16:52.25 So the other way to control malaria of course,
00:16:56.11 is to block the route of transmission.
00:16:58.25 Or at least reduce the risk of transmission. Which you can see here is a mother and her child,
00:17:04.13 sleeping under an insecticide treated bed net.
00:17:07.25 And this is a very effective way to prevent transmission.
00:17:11.15 And of course, the bed nets need to be maintained,
00:17:14.13 and the insecticide needs to be re-applied to the bed net, with a certain periodicity.
00:17:19.17 Therefore, this cannot be the only method of choice for control of the vector.
00:17:25.05 Sense leaving the responsibility of the implementation of such a public health policy to the individual,
00:17:31.13 is most likely not going to be successful or sustainable over the long run.
00:17:36.19 Now one of the primary weapons against the insect,
00:17:41.15 the vector that transmits malaria, the Anopheles mosquito, of course is insecticides.
00:17:46.12 And the most infamous insecticide of all is DDT.
00:17:50.03 DDT, or dichloro-diphenyl-trichloroethane,
00:17:54.27 is of course one of the most infamous insecticides ever applied.
00:17:59.10 And this is because, used inappropriately or irresponsibly,
00:18:03.29 for agricultural crops spraying,
00:18:06.03 can result in leakage of this compound into the environment and the ecology.
00:18:10.13 Causing detrimental effects to many species of birds in particular.
00:18:14.21 However, DDT is very cheap, very safe for humans,
00:18:18.17 and can be applied indoors, inside residents and dwellings,
00:18:23.16 where mosquitoes might land after taking a blood-meal.
00:18:26.25 And used in this conservative fashion, can prove to be a very effective weapon,
00:18:31.18 against the vector. However, because of worldwide bans against the use of DDT,
00:18:38.25 there has been decreasing use of this compound,
00:18:41.21 and the effects of decreasing use of DDT are shown here.
00:18:45.04 So in blue, are indoor residual house sprayings in South America,
00:18:49.25 as you can see, as a function of time,
00:18:52.07 going into the 1970s here, the number of dwellings sprayed decreases.
00:18:58.00 And in the red bars, shows relative number of malaria cases per year.
00:19:02.13 And so what you see is a very clear anti-correlation
00:19:05.22 between the use of DDT and the increased prevalence of the malaria parasite in South America.
00:19:13.10 And this holds true for other parts of the world as well.
00:19:16.13 And so, as a result, there are new recommendations on the part of the World Health Organization.
00:19:22.26 Basically, in 2006, the W.H.O. has said effective implementation
00:19:28.03 of indoor residual spraying with DDT, or other recommended insecticides,
00:19:32.27 should be a central part of national malaria control strategies,
00:19:36.12 where the intervention is appropriate.
00:19:39.13 And so, without the use of insecticides, we think, basically,
00:19:44.12 that the battle against malaria will be very difficult indeed.
00:19:47.12 If not totally intractable.
00:19:49.08 So to summarize, controlling malaria requires certain things.
00:19:53.23 We need a new generation of anti-malarial drugs that can be used in combination.
00:19:59.22 Again, the days of mono-therapy, single drugs, are over.
00:20:03.15 But we need combinations of cheap, effective, non-toxic, easy to make drugs.
00:20:09.11 This must be supplemented by an organized healthcare delivery system.
00:20:13.24 The infrastructure in countries that have endemic malaria
00:20:17.06 must be subsidized to be able to distribute such effective medications.
00:20:21.28 If we don't treat sick people, we do not break the chain of transmission.
00:20:26.18 Furthermore, we need sustainable vector control.
00:20:29.29 Either through insecticide treated bed nets, indoor residual spraying,
00:20:34.18 or, ecological means, such as draining swamps and so on.
00:20:39.04 This could hopefully one day be supplemented by vaccine development.
00:20:43.08 As I said before, there is no vaccine in existence today
00:20:47.04 with an operational impact against Plasmodium falciparum, or the other three Plasmodia
00:20:52.15 that cause malaria.
00:20:54.18 Yet, we can hold out hope, hopefully,
00:20:57.21 that a vaccine will be developed to help eradicate this scourge.
00:21:01.19 Without these elements, the battle against malaria will continue.
00:21:06.25 And that is going to be a big problem.
00:21:09.06 So, hopefully you have enjoyed this presentation,
00:21:12.03 and background about malaria, the disease, and it's parasites.
00:21:16.05 Thank you.
00:21:17.12

Part 2: Malaria: Research

00:00:00.00 Hi. I'm Joseph DeRisi and I'm an associate professor in the department of biochemistry and biophysics.
00:00:07.23 And I'm a Howard Hughes investigator at the University of California in San Francisco.
00:00:11.17 And it is my privilege today to tell you about our research
00:00:14.19 on the parasites that cause the human disease malaria.
00:00:18.07 So, our research goals are very simple.
00:00:22.03 If you saw my previous segment on the background and overview of malaria the disease,
00:00:26.13 you know what a devastating condition this is for humanity.
00:00:29.26 So it is our goal to develop strategies that are affordable and sustainable
00:00:34.23 for the treatment of the disease malaria.
00:00:36.25 And we are going to go about this in two different ways.
00:00:39.27 First of all, through a basic science approach using the power of genomics
00:00:44.03 to understand more about the basic molecular physiology of the parasite Plasmodium falciparum.
00:00:49.13 The causative agent of the most deadly form of human malaria.
00:00:52.17 Second of all, a more near term approach is to take a drug discovery pathway,
00:00:57.19 To find new anti-malarial chemotherapeutics for the treatment of this disease.
00:01:02.17 So, let me first review the malaria life cycle.
00:01:06.07 Shown here is the a cartoon simplification of the life cycle of Plasmodium falciparum.
00:01:11.22 It begins with the bite of a mosquito.
00:01:14.13 The anopheles mosquito
00:01:15.20 that releases sporozoites into the bloodstream.
00:01:18.07 These travel to the liver, whereupon they invade hepatocytes.
00:01:21.21 It is here that they will undergo multiple rounds of replication, and then burst forth from the liver,
00:01:26.25 in a form called merozoites.
00:01:29.07 It is this form that will then infect red blood cells in a 48-hour,
00:01:33.15 asexual cycle that will repeat itself, essentially indefinitely.
00:01:36.19 This is where all the clinical manifestations of the disease malaria occur.
00:01:41.00 Now, with some infrequent rate, some parasites will develop into a sexual form called gametocytes.
00:01:46.10 And it is these gametocytes that can be picked up by a new mosquito,
00:01:50.18 taking a blood meal. And the entire process begins again.
00:01:54.12 And so, traditionally, researchers have focused on the asexual cycle of the parasite,
00:02:00.20 because this is where all the clinical manifestations occur.
00:02:05.08 And where drugs have been developed in the past to fight this disease in patients.
00:02:10.13 The genome of Plasmodium falciparum was completely sequenced in 2002
00:02:14.15 by Sanger, Tiger, and the Stanford Genome Centers.
00:02:18.10 It provides much of the foundation that my lab uses to research this parasite.
00:02:23.12 It was found, when it was completely sequenced,
00:02:25.17 it was to contain 14 linear chromosomes covering 22.8 megabases of DNA.
00:02:31.10 This genome codes for approximately 5,400 genes.
00:02:35.23 So in comparison, this is roughly the same size genome in number of genes
00:02:40.04 as the model organism yeast, Saccharomyces cerevisiae
00:02:43.13 Of which we know so much about.
00:02:45.07 This is in contrast to Plasmodium falciparum, which we know very little.
00:02:49.07 The genome was also found to contain a 35 Kb circular plastid genome,
00:02:54.19 and a 6 Kb linear mitochondrial genome. Now, one of the most
00:02:58.15 remarkable features of this genome is its very high percentage of AT relative to GC.
00:03:05.09 And a very high coding density.
00:03:08.11 Also remarkable in this genome,
00:03:09.16 is the fact that 60% of all the genes found in the genome have essentially no known function.
00:03:15.21 This is an extraordinary high percentage.
00:03:18.01 We are sequencing more and more genomes every year.
00:03:21.00 And with that comes more and more knowledge about every gene.
00:03:24.09 Yet in Plasmodium falciparum, the vast dominant portion of the genes,
00:03:28.16 still have no known function.
00:03:30.15 Now, to understand the work that we are doing,
00:03:35.14 I'd like to tell you about the asexual blood stage.
00:03:38.03 And this is where all the clinical manifestations of the disease occurs.
00:03:41.04 It begins of the release of merozoites into the blood stream,
00:03:45.01 which then infect red blood cells.
00:03:47.07 This is the so-called ring stage of the parasite.
00:03:49.20 After the ring stage, follows a metabolic stage of the parasite called
00:03:53.18 the trophozoite phase.
00:03:55.05 This is then followed by multiple rounds of replication, and the so-called schizont phase.
00:04:00.10 After the schizont phase,
00:04:01.29 the erythrocyte ruptures, releasing merozoites,
00:04:04.12 and this 48-hour cycle repeats itself, essentially indefinitely.
00:04:08.18 And so what we really wanted to know is,
00:04:11.24 what genes are expressed during the asexual blood stage,
00:04:14.29 and when.
00:04:15.24 Why?
00:04:16.14 Because the genes that are expressed could provide clues
00:04:19.19 as to the biochemical mechanisms that are occurring during this phase.
00:04:23.07 And may highlight the pathways or targets
00:04:26.26 that can be used for anti-malarial chemotherapeutic intervention.
00:04:30.10 And so, to know more about what genes are used,
00:04:33.20 and when throughout the asexual blood phase of the parasite,
00:04:36.29 we decided to take a DNA microarray approach.
00:04:40.16 That is, we want to make a DNA glass chip on which we print
00:04:45.19 all the genes of Plasmodium falciparum,
00:04:48.11 for the purpose of measuring the expression level of all genes in the genome simultaneously.
00:04:54.12 So, a DNA-microarray is nothing but a glass slide,
00:04:57.26 on to which is printed DNA from different genes,
00:05:01.04 so each spot shown on this array here, is a different gene from the genome.
00:05:06.02 This entire chip measures only 18 millimeters across in diameter.
00:05:10.12 So we can use this as a hybridization target.
00:05:12.26 Now, if you had a very powerful microscope, like an atomic force microscope,
00:05:17.13 and zoomed in on one of these spots,
00:05:19.14 this is what you would see.
00:05:21.10 What we would see here, in this ASM microscope,
00:05:24.06 taken courtesy of Hewlett-Packard,
00:05:25.26 in the late 1990s,
00:05:27.07 is all the strands of the DNA of this microarray laying flat on the glass in a monolayer.
00:05:33.01 So this provides an excellent hybridization target,
00:05:35.23 for subsequent assays like gene expression monitoring.
00:05:39.09 Now, how are these chips made, and how do we go about this process?
00:05:43.06 So, what we did is we built machines in the lab
00:05:46.29 that can make these so-called DNA-microarrays,
00:05:49.02 these microarray printers are easily constructed,
00:05:51.29 and can be built from simple directions.
00:05:53.28 And so if you are so inclined, I invite you to go visit our webpage at
00:05:57.07 derisilab.ucsf.edu/core
00:05:59.29 and download the directions for building your own DNA-microarray printer.
00:06:04.05 This would enable labs to print essentially unlimited chips
00:06:08.13 on their own time in their own labs,
00:06:10.02 without having to rely on commercial sources for such reagents.
00:06:14.02 So how does a typical microarray experiment actually occur?
00:06:18.23 So, with respect to malaria, an experiment might go down something like this.
00:06:23.14 You start with two different phases of the blood phase
00:06:28.10 So here, this is an early trophozoite, and this is a late schizont.
00:06:33.03 You can isolate parasites from each of these phases,
00:06:36.05 by growing them in a synchronous fusion.
00:06:38.10 You can then isolate their RNA, which is the manifestation of their DNA,
00:06:42.15 the gene expression that occurs,
00:06:44.22 converted to cDNA using reverse-transcriptase.
00:06:47.14 And then labeled, the subsequent cDNA, with fluorescent dyes.
00:06:52.07 You can label one in red, and one in green.
00:06:54.18 And then we can co-hybridize that to the array.
00:06:57.13 And so, what we see is the relative abundance level of each gene expressed
00:07:02.12 as a ratio of colors on each spot, between the red and the green.
00:07:06.04 And this is how a typical microarray expression works.
00:07:09.15 But, there is one complication:
00:07:11.28 I don't simply want to measure the difference between a trophozoite and a schizont.
00:07:16.12 What I'd like to do is measure the gene expression levels of every gene in the genome,
00:07:20.13 every hour, throughout the 48-hour life-cycle,
00:07:23.07 to capture the dynamic nature of the transcriptional network as it plays out in this parasite.
00:07:29.02 And so, what we decided to do is isolate RNA from each stage,
00:07:34.09 every single hour, for two days essentially.
00:07:36.23 And to do this, we had to grow a very large culture of parasites.
00:07:40.10 So we utilized a 5-liter bioreactor system
00:07:43.13 that we could fill with human blood harvested from volunteers,
00:07:47.14 infect with parasites,
00:07:49.13 and then keep in very controlled conditions:
00:07:51.29 temperature, pH, gas, agitation, and so on.
00:07:56.03 And so this synchronous culture, growing as shown here,
00:08:01.10 we were able to take out samples on the hour, every hour.
00:08:04.13 And this shows the relative synchrony of the parasites as they are growing in this fashion.
00:08:08.23 And so, what did we see when we did this experiment?
00:08:13.06 We pulled out RNA every hour, on the hour,
00:08:15.15 we hybridized it to our DNA microarrays
00:08:17.22 that represents the whole Plasmodium falciparum genome,
00:08:20.12 and we took a look at some genes.
00:08:22.11 So, here is an example gene.
00:08:24.03 This is a gene called erythrocyte binding antigen 175
00:08:27.26 , or EBA175, a very well characterized gene in Plasmodium falciparum.
00:08:32.12 And what we can see is, early in the cycle, there is very high expression,
00:08:36.20 left over from the late schizonts as they are releasing merozoites,
00:08:41.16 and then expression just continues to crash out,
00:08:44.19 and it reaches a low point during the trophozoite phase,
00:08:47.12 and then immediately rockets up and reaches its highest point,
00:08:51.11 during the point of invasion.
00:08:53.08 When merozoites are again being made and released into the blood stream.
00:08:56.17 Now Peak to trough, this gene exhibits a 256-fold regulation.
00:09:01.29 And this is typical of invasion type genes.
00:09:05.04 Now what we also notice, is if we looked at other genes in the Plasmodium falciparum genome,
00:09:09.14 and looked at their expression profile,
00:09:11.20 we noted that they almost all have one trough, and one peak.
00:09:16.03 That is, most genes in the genome that are expressed at decently high levels,
00:09:21.08 throughout the asexual lifecycle, exhibit periodic expression.
00:09:26.10 They are induced and repressed approximately only once per cycle.
00:09:30.25 So, that enabled us to use some new tools to analyze this expression.
00:09:36.14 What we did, is we took all the expression profiles,
00:09:39.25 and did a fast Fourier transform
00:09:41.16 to understand how periodic these signals are from each gene.
00:09:47.04 And what this histogram with this power specter is showing you
00:09:50.13 is that most genes are very periodic.
00:09:53.19 If we randomized the data and recalculated the fast Fourier transform with permuted profiles,
00:10:00.05 we get a distribution that is like this, which is very different from this.
00:10:03.23 So this indicates, that by and large, most genes in the genome have a wave-like expression profile
00:10:11.05 throughout the asexual blood cycle.
00:10:13.11 So, if we then extract the phase of expression from the fast fourier transform
00:10:17.22 of all these gene expression profiles, and plot the expression by phase,
00:10:21.20 we end up with something like this.
00:10:23.25 So shown here, are all of the genes that have measurable expression levels
00:10:29.20 throughout the asexual blood cycle on the Y axis,
00:10:32.15 approximately 2,700 genes. And across the X axis,
00:10:36.09 are all the time points from 0-48 hours.
00:10:39.19 And what you can see here, by this red stripe that proceeds down this way,
00:10:44.15 is a very smooth cascade of expression.
00:10:46.28 That is, the genes are being induced once and only once per cycle,
00:10:50.27 in a clean cascade that starts with the beginning of the ring stage,
00:10:56.06 ends with the schizont stage, and then wraps around to the beginning.
00:11:00.10 So, what we observed in this very smooth cascade of gene transcription during the asexual
00:11:08.10 cell cycle of the Plasmodium falciparum parasite,
00:11:11.03 was a certain metabolic logic to the genes induced and when they were induced.
00:11:15.15 So if we take a closer look at this logic,
00:11:17.19 we can basically understand what the parasite is doing during this time.
00:11:22.15 So, beginning with invasion,
00:11:25.10 we see genes immediately induced that have to do with both building ribosomes,
00:11:29.16 translation machinery, and transcription complexes.
00:11:32.18 This is intuitive because these are the building blocks of all eucaryotic cells,
00:11:36.21 and are required to make more of the same.
00:11:39.12 Now following that induction of these genes,
00:11:42.06 what we see are genes involved in both glycolysis,
00:11:46.28 the main energy production system of the cell,
00:11:49.12 and ribonucleotide synthesis.
00:11:51.13 That is, making more substrates to make more RNA, the manifestation of genes themselves.
00:11:56.19 This is then followed with some phase delay,
00:11:59.14 by genes involved in making deoxyribonucleotides. The building blocks of DNA.
00:12:04.19 And not surprisingly, we see that this is directly in phase
00:12:07.22 with genes involved in DNA replication.
00:12:10.05 Now, I wonder if you can basically see this here,
00:12:13.10 but it appears as though the genes and when they are induced, resemble a just in time production process.
00:12:19.27 That things are made when they're needed, in the amount they are needed, and then they are shut off.
00:12:24.15 This is how many commercial car factories work.
00:12:28.02 Now, one of the problems with this, is a supply of a certain part doesn't get there in time,
00:12:33.05 or just in time. And this can cause, to, errors during the system,
00:12:37.22 or collapse of the whole system.
00:12:39.14 After this point in the cascade, we see genes induced that have to do with the TCA cycle,
00:12:45.07 and the proteosome, and then quickly following that,
00:12:48.06 we find that genes are induced that have functions related to plastid,
00:12:52.28 and merozoite formation.
00:12:54.28 So what we can see here is the beginning of the process of building new parasites
00:12:58.26 for reinvasion of new cells.
00:13:00.17 This concludes with, genes explicitly involved in the invasion phase.
00:13:06.13 Actin-myosin motility genes,
00:13:08.08 are known to be involved in the process of physically invading new erythrocytes.
00:13:13.00 And we see these in genes induced right at the end of the cycle.
00:13:16.23 And this finally concludes with early ring transcripts,
00:13:20.09 that is beginning the whole cycle all over again, with newly infected erythrocytes.
00:13:25.00 And so what I hope I've shown you here, is that Plasmodium falciparum gene expression,
00:13:30.16 appears as a single cascade that proceeds throughout the 48 hours,
00:13:35.03 and represents a certain metabolic logic,
00:13:37.22 reminiscent of a just-in-time production facility.
00:13:40.07 Now, was this just a fluke?
00:13:42.29 Is this just the way Plasmodium falciparum looks like in the lab, in this one particular strain?
00:13:48.08 We sought to replicate this experiment
00:13:50.03 two more time with different strains of Plasmodium falciparum, that come from different parts of the world.
00:13:55.14 And shown here, are all three side by side.
00:13:58.08 And all though there are small differences in the synchrony of the parasites,
00:14:01.26 What I think that you can see quite clearly here is that,
00:14:05.01 for all practical purposes, all three transcriptomes are nearly identical.
00:14:09.29 That is, it appears as if Plasmodium, from different parts of the world,
00:14:15.03 express exactly the same transcriptome cascade as their relatives in other parts of the world.
00:14:21.29 And so, what this leaves us with, is many outstanding questions.
00:14:27.03 Like all very interesting discovery based science,
00:14:30.08 more questions are generated than answered.
00:14:32.08 And this leads us to the next slide, which will discuss these.
00:14:36.00 To what extent is the transcriptome, that we observed, rigid?
00:14:39.08 How do we know that there are response genes,
00:14:42.08 genes that can change this transcriptome, in relation to environmental perturbations?
00:14:46.16 It's been found by others in the field,
00:14:49.27 that very few transcription factors are coded for in the genome.
00:14:53.04 At least apparent transcription factors that
00:14:55.11 are recognizable using common bionformatic techniques.
00:14:58.04 So could it be that Plasmodium falciparum possesses a very streamlined,
00:15:03.19 stripped-down, transcriptional complement?
00:15:07.25 It's possible.
00:15:08.28 What are the cis-acting determinants of the expression phase?
00:15:12.05 It is known that the cis-acting region upstream of genes determine their phase of expression.
00:15:18.11 But we don't know what are the elements inside
00:15:21.01 these promoter regions that are responsible for driving phase of expression.
00:15:25.11 And, a very related question,
00:15:27.25 what are the transcription factors that bind those putative cis-acting elements?
00:15:31.28 And what are their roles in each of the phase-specific expression profiles?
00:15:35.29 To date, very little work has been done on transcription factor biology in Plasmodium falciparum.
00:15:41.23 Yet, it is quite clear that if one can design an anti-malarial chemotherapeutic
00:15:46.22 to disrupt the regulatory process, because the transcriptome appears to be rather rigid
00:15:53.06 in its style of expression. This could potentially result in catastrophe for the parasite.
00:15:58.28 That is, perhaps this apparent rigid transcriptome is the Achilles heal of Plasmodium falciparum.
00:16:06.05 And anything that we do that would misregulte, or cause a perturbation,
00:16:12.05 to the regulatory cascade as we observe it, may kill the parasite easily.
00:16:17.04 And that brings us to our final question:
00:16:18.27 Can we disrupt this transcriptome?
00:16:21.10 Now, if you are interested in the data that I've told you about today,
00:16:24.11 and you'd like to browse, either the whole genome, or gene by gene,
00:16:27.27 all the data which I’ve showed is available on our website at malaria.ucsf.edu.
00:16:33.16 And I encourage you to log on, pick out your favorite genes,
00:16:36.20 or just browse the genome for functions and expression profiles.
00:16:40.00 And try to understand basically what the parasite is doing.
00:16:44.00 Many, many projects can emanate from such data, and I encourage you to take a look.
00:16:48.06 Thank you.
00:16:50.11

Part 3: Malaria: Drug Development

00:00:00.00 Hi, my name is Joseph DeRisi, and I'm an associate professor at the University of California,
00:00:07.20 San Francisco in the department of Biochemistry and Biophysics,
00:00:10.23 and I'm a Howard Hughes investigator.
00:00:12.24 I'll be giving you a lecture today about or labs efforts
00:00:15.12 to develop drugs for the disease of malaria.
00:00:18.05 So first, a little background on the disease itself.
00:00:22.15 It's a horrible disease caused by parasitic protozoa.
00:00:25.03 It is characterized by intense fevers, chills, sweats, and in its worst form,
00:00:29.15 cerebral malaria can cause death.
00:00:31.25 Why do we care about the disease?
00:00:33.12 Why is there a need to develop drugs?
00:00:35.10 Very simple. The disease exacts an intolerable toll upon humanity.
00:00:40.15 Somewhere between one and two million deaths per year.
00:00:43.13 That is someone who dies every 30 seconds.
00:00:46.00 Furthermore, between 400 and 900 million people will come down with the disease per year.
00:00:52.15 This causes a huge socioeconomic burden anywhere there is endemic malaria.
00:00:57.05 So, why is there a problem with this disease? Well, first of all, there is world-wide drug resistance.
00:01:03.25 The limited drugs that we have, and I'll be reviewing those in a second,
00:01:06.27 have become ineffectual in many parts of the world.
00:01:11.09 Furthermore, we have no vaccine to date with an operational impact.
00:01:15.19 And, because people who live in endemic regions only acquire limited immunity,
00:01:21.26 and that limited immunity requires repeat infections,
00:01:25.07 we have only a limited capacity to ultimately control this disease.
00:01:30.17 OK. So, first of all,
00:01:34.09 why is there not major large drug efforts in malaria by the major pharmaceutical companies?
00:01:41.09 There are some limited efforts,
00:01:42.29 but for the most part, malaria occurs in the poorest regions of the world,
00:01:47.04 therefore, the economic incentive to develop drugs for malaria
00:01:51.16 is not nearly what the economic incentive it is to, say, develop drugs for obesity,
00:01:56.11 or hair loss. Now, this disease is also transmitted by a vector,
00:02:01.00 the anopheles mosquito. And this complicates issues in controlling the disease.
00:02:05.09 So, the disease itself, malaria is caused by one of four parasites that infect humans:
00:02:11.29 Plasmodium falciparum, the one that we study,
00:02:14.27 and it is in fact the one that causes the most deaths and disease throughout the world.
00:02:19.10 Plasmodium vivax, Plasmodium malariae, Plasmodium ovale.
00:02:23.10 These are all important parasites. But falciparum is considered to be the scourge of humanity.
00:02:28.11 This, is a blood smear of malaria.
00:02:31.06 And basically, if you took blood from an infected individual and stained it with a stain called giemsa,
00:02:36.15 that highlights where DNA is in cells, you can see that this cell here, and this cell here,
00:02:42.09 are infected with parasites. Whereas these healthy red blood cells here and here are uninfected.
00:02:47.26 It is these parasites in the blood phase that we wish to develop therapeutics for, to kill.
00:02:53.02 So, quickly, let's review the whole life cycle.
00:02:55.23 Infection by malaria begins with the bite of a mosquito.
00:02:59.27 Mosquitoes release from their salivary glands sporozoites,
00:03:03.12 which travel through the blood stream, into the liver.
00:03:06.00 It is here that they infect hepatocytes.
00:03:08.14 After going through multiple rounds of replication,
00:03:11.22 these parasites will burst forth from the liver in a form called merozoites,
00:03:15.15 and begin to infect a cycle of red blood cells.
00:03:18.19 The parasites invade the red blood cell, consume the hemoglobin within
00:03:22.18 as an amino acid source, and then replicate in multiple rounds, and burst forth again.
00:03:28.12 And this process repeats itself, again and again, again. essentially in perpetuity,
00:03:32.29 unless the immune system brings the infection under control,
00:03:35.17 or the victim succumbs to the infection.
00:03:37.22 Now, with some irregular frequency, parasites will develop into a sexual form,
00:03:42.11 called gametocytes. And it is this form that will be taken up by a new mosquito
00:03:46.23 at a blood meal, and the entire cycle repeats again.
00:03:49.28 So clearly, there are several different places which malaria can be controlled.
00:03:54.02 Malaria can be controlled primarily by wiping out the mosquito.
00:03:58.17 That is, vector control can take care of most of the problems of malaria,
00:04:03.19 if you have the money, the geography to support it, and the social structures to support it.
00:04:08.09 Vaccines, seek to try to block the initial infection of the sporozoite in to the liver.
00:04:14.06 But as I said, so far, there is no vaccine with an operational impact.
00:04:18.15 Perhaps one day, but we don't have it right now.
00:04:21.10 To date, all the success in controlling malaria, the disease in people,
00:04:26.13 has been with chemotherapeutics, drugs.
00:04:29.06 And most drugs target this asexual erythrocytic phase of development of the parasite.
00:04:34.13 So, let's quickly review malaria drugs as they exist today.
00:04:41.18 Now, the most famous of all drugs is quinine.
00:04:45.08 Quinine was isolated from the bark of a tree, and was used by native south Americans,
00:04:52.26 and the Jesuits through the 1600s on.
00:04:55.28 It is one of the most famous of all drugs because of its amazing potency against malaria.
00:05:02.28 Now, one of the most next famous drugs is chloroquine.
00:05:05.20 Chloroquine was developed originally by German pharmaceutical chemists,
00:05:11.10 but then taken over by American pharmaceutical chemists during world war 2.
00:05:15.11 And, after world war 2, became the drug of choice.
00:05:18.13 Now this was a wonderful drug. This drug was relatively non-toxic,
00:05:22.25 it was very cheap to make, and had an extremely good potency.
00:05:27.22 Yet, the drug was misused.
00:05:29.20 During the 1950s and 60s, there was irresponsible overuse of this drug.
00:05:34.07 And as we know, when we overuse a single drug against a single target or pathogen,
00:05:39.12 resistance can develop to that drug. And that is exactly what happen.
00:05:42.29 So in many parts of the world today, chloroquine is very ineffectual.
00:05:48.08 And so we need new drugs to bring a new generation
00:05:52.14 of chemotherapeutics to bear on this problem.
00:05:55.02 Now, after chloroquine, came mefloquine was also developed by Walter Reed, the U.S. Army.
00:06:00.20 And this was in response, in part, to chloroquine having resistance problems.
00:06:06.00 Although mefloquine is a very expensive drug to make,
00:06:09.12 and in a certain percentage of people causes fairly severe neurological side-effects.
00:06:14.04 So, this Vietnam-era drug cannot be considered to be
00:06:18.20 a cheap widespread chemotherapeutic for malaria control
00:06:22.11 Now, after this, halofantrine, halofantrine is again a U.S. Army drug,
00:06:27.23 and halofantrine was meant to overcome problems of mefloquine and chloroquine resistance.
00:06:32.19 This drug, also developed by Walter Reed, suffers from certain cardio toxicity side-effects.
00:06:38.06 Now, in the 1990s, we came to realize that artemisinin,
00:06:43.19 a drug originally derived from Chinese herbal medicine, is probably one of our best hopes.
00:06:49.17 Now there are side-effects with this drug as well, Although, currently,
00:06:53.15 there has not been very much in the way of resistance described for this drug.
00:06:57.29 So, how do we go about developing new drugs?
00:07:04.01 And what are the considerations we must take into account when developing them?
00:07:08.12 Well first of all, the cost of goods must be cheap.
00:07:11.14 If we are going to make a drug for malaria,
00:07:13.06 we have to shoot for a target of about ten cents a curative dose.
00:07:16.20 Any higher than this, and the populations that need it will not be able to afford it.
00:07:20.22 Furthermore, the time to resistance of new compounds must be slow.
00:07:25.05 The money and effort that we put into drug development,
00:07:27.10 only to have the compound become useless in the field,
00:07:30.08 very rapidly, is obviously not a very good effect.
00:07:33.26 The other thing is, side-effects must be minimal.
00:07:36.21 So, toxicity profiles are extremely important.
00:07:40.18 And first of all, and most importantly,
00:07:43.23 we must realize that the era of monotherapy is completely over.
00:07:47.26 We know from HIV that treating with a single drug only breeds resistance quickly.
00:07:53.02 And if we use drugs in combination, together,
00:07:55.25 to target different areas of the pathogen, resistance is much slower to arise.
00:08:01.21 And so, we don't just need one new drug,
00:08:04.25 we need several new drugs, and we need to develop them in combinations.
00:08:08.13 So, my lab is taking two different approaches to anti-malarial drug development.
00:08:14.29 There are many labs and companies around the world that are developing malaria drugs.
00:08:19.18 Although, not as many as I'd like to see personally.
00:08:22.24 And so I'm only going to be describing our small efforts
00:08:26.00 to develop anti-malaria chemotherapeutics.
00:08:28.21 There are two approaches I'd like to tell you about.
00:08:31.05 So the first is a Parallel Synthetic Chemistry approach.
00:08:34.20 The so-called Privileged Scaffold approach.
00:08:36.20 And the second is another approach, called diversity screening.
00:08:41.24 That is, why don't we look for new anti-malarials amongst known drugs.
00:08:46.05 But first, before I tell you about these two efforts,
00:08:49.05 I have to tell you about the assay that we use.
00:08:51.15 Any drug-screening effort, or drug-testing effort always begins with an assay.
00:08:56.15 And the assay here is a growth assay.
00:08:59.22 Now as I told you, the intra-erythrocytic cell cycle of malaria is what we target with most drugs.
00:09:05.22 During the blood phase of malaria infection,
00:09:08.01 this is where all the clinical manifestations of the disease actually occur.
00:09:11.27 So this is the phase we want to target.
00:09:14.16 It's a 48-hour cycle, it begins with infection as I mentioned before by merozoites.
00:09:18.06 These merozoites develop into the ring-stage inside the erythrocyte.
00:09:22.02 There is a main metabolic phase in which the parasite grows.
00:09:24.27 Multiple rounds of replication occur.
00:09:27.04 And then these little parasites burst out and this cycle begins again.
00:09:30.09 And so what we do, is we treat parasites beginning here in this phase,
00:09:35.13 we let them go all the way through this cycle, past merozoites, and into the next cycle.
00:09:40.28 72 hours. And what we measure, is the increase in the number of infected cells over this 72 hours.
00:09:48.07 A good drug, if it kills parasites won't go around this cycle,
00:09:52.10 and therefore we will not see an increase in the number of infected cells.
00:09:55.24 So that's our goal. How do we measure the number of infected cells?
00:09:59.26 Well you could stain cells and count them, much the way these cells are stained here.
00:10:03.26 But that would be laborious, time consuming,
00:10:06.02 and if you didn't count a lot of cells, it could be statistically inaccurate.
00:10:10.02 So we use a flow-cytometer.
00:10:12.28 A flow-cytometer is nothing more than basically a fancy fluorescent microscope
00:10:17.07 in which cells past the objective of the microscope one at a time very fast.
00:10:21.12 In this case, about 30 thousand cells per second.
00:10:24.00 And this microscope has the ability to count
00:10:26.13 and measure the fluorescence in each cell as it passes the objective.
00:10:29.14 What we do is stain cells with a fluorescent dye. erythrocytes, blood cells,
00:10:36.02 have no nuclei and no DNA when they are mature.
00:10:39.23 Parasitized red blood cells have DNA. So if we stain with a fluorescent dye,
00:10:45.16 those cells will be fluorescent. And the flow-cytometer will be able to count those
00:10:49.10 and differentiate those from non-infected cells.
00:10:52.19 So, if we look at the data from the flow cytometer, here is a graph of uninfected cells.
00:10:57.18 This is a histogram, this is the number of cells, on this axis, and this is the fluorescence.
00:11:03.02 And so here is all the red blood cells with minimal fluorescence.
00:11:06.17 Here is an infected blood cell population.
00:11:08.25 So you can see here, here is the non-infected cells,
00:11:12.06 and then here is the infected population. And you can clearly separate these two peaks.
00:11:17.00 These are the cells here that we actually want to count.
00:11:20.03 And we can just count those cells up as a proportion of the total,
00:11:23.09 and we will get an absolute number of the parasitemia.
00:11:26.01 That is, the percentage of cells that are infected.
00:11:28.29 And what we want to then do, is just measure how many cells,
00:11:32.23 before we treated with the drug, and after we treated with the drug, relative to a control with no drug.
00:11:38.03 Now, we don't put in tubes of these cells one at a time,
00:11:42.13 because we wanted to actually do thousands of tubes,
00:11:44.21 So we developed a small program to drive a robot
00:11:47.29 that actually feeds the cells to the machine in an automatic fashion.
00:11:51.28 So, the little robot is shown here.
00:11:55.09 And all the cultures that we do are in 96-well plates,
00:11:58.07 and this little sipper will come and get cells from each of these wells, and inject it into the machine here.
00:12:04.07 So, let's watch a little movie of this in action.
00:12:07.11 So, what you can see here is the machine beginning to take up cells from each plate,
00:12:13.06 and inject it, here. And it goes into a loop, and into the flow-cytometer.
00:12:17.10 And while those cells are being counted, this machine goes and gets another well and injects it in,
00:12:21.27 and this repeats over and over again.
00:12:23.08 So, it takes about 12-13 minutes to complete an entire 96 well plate.
00:12:28.19 And we can have several 96-well plates on deck to process.
00:12:33.09 So it makes the process of counting multiple replicates of drug treatments,
00:12:38.16 very fast, very accurate.
00:12:41.02 Alright, so now that I've told you about the assay that we use, I want to tell you about our approach.
00:12:46.11 So the first approach I'm going to tell you is the so-called privileged scaffold approach.
00:12:50.29 And this entire project is a close collaboration between my lab and Kip Guy at St. Jude,
00:12:56.06 and a very talented graduate student, Peter Madrid.
00:12:59.06 So the privileged Scaffold is very simple.
00:13:01.23 I've told you that chloroquine is one of the most famous drugs, extremely potent in its day,
00:13:06.21 very non-toxic, extremely cheap to make, excellent drug overall.
00:13:12.09 So, who is to say, that the scaffold that chloroquine is built on,
00:13:16.19 the so-called four amino quinolines, is a scaffold not worth pursuing further?
00:13:22.11 That is, there may be derivatives of chloroquine, modifications of the basic scaffold
00:13:27.12 design that could produce drugs that are just as effective.
00:13:31.03 And so, if we just take a generalized structure of the quiniline series of compounds,
00:13:36.25 and we begin to systematically modify the quiniline structures
00:13:41.03 in new ways that haven't been reported before,
00:13:43.26 perhaps we can find new activities that will rival those of chloroquine.
00:13:48.06 That's the hope of course.
00:13:50.07 And so, what Peter Madrid did, is he started out with the basic scaffold,
00:13:55.24 and he decided to derivitize, that is put on different side chains and what not.
00:14:00.27 Onto different parts of the molecule.
00:14:02.22 So in this first example, what Peter Madrid did,
00:14:06.00 Is he changed these two groups here at this distal amine,
00:14:09.12 and a typical library that he made is shown here.
00:14:13.13 So, in this example of one para-synthetic library,
00:14:16.15 Peter has kept one of the groups on this distal amine constant, a propyl group,
00:14:21.04 and he has then substituted the other position on this distal amine with a variety of different side chains.
00:14:26.13 And it is these different side chains that we want to test for efficacy against the parasite.
00:14:31.21 And so, a typical activity assay looks like this.
00:14:35.09 Now what I'm showing you hear is the different activities of all these different side chains
00:14:39.06 as a function of how well they kill the parasite.
00:14:42.05 And a dark blue means they are killing the parasite very well.
00:14:45.21 So you can see, there are two different rows here.
00:14:48.08 The top row is a drug sensitive parasite.
00:14:51.05 The bottom row is a drug resistant parasite that is known to be resistant to chloroquine.
00:14:56.25 So you can see, here is our chloroquine control over here,
00:14:59.09 and you see only a dark square at the top, not at the bottom.
00:15:02.11 And you can see that both compounds work just as well against both strains of malaria,
00:15:07.08 whereas some compounds, such as these two over here,
00:15:10.11 only work against the drug sensitive version of malaria.
00:15:13.23 And so obviously, we will take the compounds that work against both,
00:15:17.24 move them to the next phase, and then begin to try different side chains
00:15:21.27 at different parts of the quiniline structure,
00:15:23.29 to in a systematic way, iteratively optimize the compound.
00:15:28.03 And so in this manner, we can create large numbers of compounds,
00:15:33.04 each individually synthesized, and optimized over several different libraries
00:15:38.08 for the best efficacy against drug-resistant and drug-sensitive parasites.
00:15:43.00 So, one of the key aspects of this project is,
00:15:47.13 we don't want to waste money continuing on drugs that are never going to work.
00:15:51.07 So, it is the philosophy of fail early, and fail cheap.
00:15:55.04 And we have help from SRI on this with John Marcellas.
00:15:58.18 Basically, we will test compounds for things like solubility,
00:16:02.18 some of the most easy tests. genotoxicity using the so called Ames 2 test.
00:16:07.16 Cytochrome P450 in addition, to test to see whether these drugs will interfere
00:16:11.23 with the metabolism of other drugs in humans.
00:16:14.13 Permeability. This is a measure of how well the drug might be taken up by cells.
00:16:18.28 This is a proxy for measuring basically bio-availability.
00:16:22.19 Will this drug be useful as an oral drug, for example?
00:16:27.09 The, we also want to know how well the drug partitions into red blood cells
00:16:31.18 versus serum. This is important.
00:16:33.23 Because in malaria, the parasites are in the red blood cells.
00:16:36.18 We want the drug to preferentially be taken up in the erythrocyte relative to the serum.
00:16:41.22 This is one of the beneficial characteristics of chloroquine.
00:16:44.14 Now, one of the most important things to look at is stability.
00:16:48.08 Taking human liver microsomes, they can be subjected to the compound,
00:16:52.20 and we can calculate the half-life of the compound when subjected to these liver microsomes.
00:16:58.18 These are basically, you know, collection of liver enzymes
00:17:02.02 that will metabolize a broad range of compounds.
00:17:04.21 A compound that only hangs around for a couple of minutes is not going to be very useful.
00:17:10.01 Chloroquine on the other hand, the model for these kinds of drugs, has a very long half life.
00:17:14.23 This is important, because when you give a dose of a drug to a patient,
00:17:18.28 you want enough compound to stay around in the body,
00:17:21.15 to have an effect on killing that parasite,
00:17:24.10 without having to re-dose that patient.
00:17:26.23 Finally, a more expensive assay is the rat oral pharmacokinetics.
00:17:31.18 How well does it get into the blood?
00:17:33.20 Is it orally available?
00:17:35.04 Does it have any toxicity against other organs, kidneys and so on.
00:17:38.27 And so, by these sort of pre-clinical measures, we can take these compounds,
00:17:43.21 and quickly weed out the ones that are never going to move forward,
00:17:46.16 because they have bad characteristics.
00:17:48.02 And the ones that pass all these tests,
00:17:50.10 they can move forward to the next iterative round of para-synthetic chemistry.
00:17:54.23 Now, where are we today?
00:17:57.20 So Peter Madrid came up with these top four compounds
00:18:00.20 that have gone through several rounds of pre-clinical testing, and iterative drug design.
00:18:04.26 And these compounds all have very similar styles of scaffold, as you can see.
00:18:10.15 And all are extremely effective against both drug-resistant and drug-sensitive parasites.
00:18:15.25 And they all have very decent half-lives. So obviously,
00:18:19.21 the next step of these compounds is to get them into more advanced pre-clinical trials,
00:18:23.26 like studying cardio-toxicity. And studying malaria infection in a primate model.
00:18:29.17 If the compounds can pass these hurdles, if any one of the compounds passes these hurdles,
00:18:34.26 they'll be moved forward.
00:18:36.09 Ultimately, to phase one human clinical trials.
00:18:39.01 That's our hope. Most drugs never make it there.
00:18:42.06 Many compounds come in, few come out.
00:18:44.19 This is the rule, but if we don't try these different compounds,
00:18:47.25 if we don't pursue the different variations on the quiniline scaffold,
00:18:51.03 we will obviously never have a success.
00:18:53.09 OK, so, basically that is one approach.
00:18:57.28 The other approach is to short-cut the drug development process.
00:19:01.21 How can we take collection of known bio-active, or approved drugs,
00:19:06.24 and mine them for anti-parasitic activities?
00:19:11.05 That is, maybe there are drugs in use today that are already good against malaria.
00:19:15.10 That have already been validated for toxicity profiles.
00:19:18.10 That are already known to be cheap and easy to make. Maybe they are generic drugs.
00:19:22.05 So, the goal is very simple. Acquire libraries of known bio-active, or approved drugs,
00:19:27.17 and screen them for malaria.
00:19:29.00 So this is the project of Jenny Weisman in my lab, in collaboration with Kip Guy.
00:19:34.17 And essentially, what she took, is a collection of about 2,000 different compounds
00:19:39.18 that are bioactive or known to be approved drugs,
00:19:43.09 and screened them for malaria activity.
00:19:45.12 And this resulted in a number of different classes.
00:19:48.22 First of all, we found a number of compounds in the collection
00:19:52.20 that are already known anti-malarial drugs.
00:19:55.01 We are not interested in these because people have already studied them,
00:19:59.10 we already know they are anti-malarial potential.
00:20:02.21 Now, there are a bunch of novel compounds
00:20:05.02 that were not previously known to have anti-malaria activity.
00:20:07.26 These are especially interesting to us. We can break these into three categories.
00:20:12.01 First of all, the oral drugs. This is important, because if we want to develop a pill-like drug,
00:20:18.13 it has to be orally bio-available.
00:20:20.02 The I.V. drugs, intravenous administered drugs, and topical drugs.
00:20:25.00 Now, topical drugs are not very interesting to us,
00:20:28.03 those are not going to be able to get into the blood stream, and so on, so we did not pursue these further.
00:20:32.24 Intravenous drugs could be interesting, but they are not our highest priority.
00:20:36.29 Oral drugs are obviously the way to go here,
00:20:39.12 if we want to develop a cheap therapeutic that can be taken in pill-like form.
00:20:43.15 These broke down into several different categories,
00:20:47.00 which I'll just highlight for you here on the screen.
00:20:49.04 All of these, the ones that we chose from the oral category,
00:20:52.15 had high therapeutic index, that is, the concentration that they kill malaria at,
00:20:57.14 was similar to the concentration that they are used at for their normal clinical purposes.
00:21:02.13 And what we have here is a bunch of different compounds
00:21:06.00 that really have very different normal uses.
00:21:09.15 So propafenone is an anti-arrhythmic drug for heart conditions,
00:21:14.18 perhexilene is a vasodilator, and these here, are actually anti-psychotic drugs.
00:21:20.09 Now propafenone is probably the most interesting drug to us,
00:21:23.17 because it is thought to either calcium, potassium, or sodium channels.
00:21:28.08 It is not clear which, so we are very interested to see if propafenone is blocking a parasitic channel.
00:21:34.05 And so more work will be done validating the target of this drug,
00:21:37.27 and developing new versions of the propafenone scaffold to test it against our model.
00:21:43.08 That is, perhaps we can increase the efficacy of this drug,
00:21:46.13 and decrease its potential effects on the human channels.
00:21:49.18 That is the hope, and of course that is going to take several years of Para synthetic chemistry,
00:21:55.00 and structure activity relationship.
00:21:57.05 OK, so our future directions here are quite clear.
00:22:01.00 Basically, we want to engage in additional rounds of structural activity relationship,
00:22:05.15 in both the quiniline series, and the propafelone series.
00:22:09.08 We want to screen additional bioactive compounds.
00:22:11.24 There are hundreds of thousands of compounds out there that are known to have biological effects.
00:22:16.17 We would like to mine them all for potential anti-malarial activities.
00:22:19.29 We would like to validate and discover new targets.
00:22:22.26 In many of these cases, like propafenone, we don't know what the parasitic target is.
00:22:26.23 Validating the target would help us understand the drug,
00:22:30.06 how it works, and potentially, how to make the drug work better.
00:22:34.22 Now, we would also like to investigate possible avenues of resistance.
00:22:38.17 We know drug resistance can develop, it developed for chloroquine.
00:22:41.26 It might develop for our new quinilines.
00:22:43.17 How do we know what the resistance mutations will be if we,
00:22:48.24 in a theoretical or hypothetical situation over use the drug?
00:22:53.03 This is of primary importance.
00:22:55.04 Also, we would like to expand testing to multi-drug resistant parasites.
00:22:59.07 The parasites that we use in the lab today may not be reflective of what is in the field today.
00:23:04.00 So bringing back parasites from sub-Saharan Africa or south-east Asia or south America,
00:23:09.08 to test in the lab, against our compounds is of critical importance.
00:23:13.14 And furthermore, for our advanced compounds, we would like to complete pre-clinical testing,
00:23:18.08 and try to drive these compounds forward to the drug development process,
00:23:21.29 Ultimately, in our best hopes, into phase 1 human clinical trials.
00:23:26.19 Of course, this is a long arduous road, and it is very expensive.
00:23:30.21 But, because academic institutions do not have the same profit incentives,
00:23:35.12 or profit motives that large pharmaceutical companies do,
00:23:38.15 we have the freedom to explore these different avenues.
00:23:41.16 To take early leads and late leads and explore them.
00:23:44.22 Without the need to answer to stock holders, for example.
00:23:47.21 So, if you are in academic science and you're interested in malaria drug development,
00:23:51.27 I hope I've showed you today that this process is actually very straight forward.
00:23:55.25 And that anyone, with a moderately sized budget and lab can get involved in the drug development process
00:24:02.03 without the need of major pharmaceutical backing.
00:24:04.28 And so, with that, I concludes this section on malaria drug development.
00:24:09.09 Thank you very much.
00:24:10.20

Videos in this Talk

Part 1: Malaria: The Disease and Parasites
Audience:
- General Public
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: Malaria: Research
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 3: Malaria: Drug Development
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Joseph DeRisi

Total Duration: 1:02:24

Recorded: June 2006

All Talks in Microbiology

Talk Overview

This brief set of three lectures gives a very general overview of malaria, the disease and Plasmodium falciparum, the causative agent of the most deadly form. Basic research as well as drug development efforts will also be covered in parts two and three of this series.

Speaker Bio

Joseph DeRisi

Joseph DeRisi, PhD., is currently a professor at UC San Francisco in the department of Biochemistry and Biophysics and a Howard Hughes Medical Institute Investigator. Prior to joining the faculty at UCSF in 1999, Joe was a UC Fellow for approximately one year. During his graduate work at Stanford University in the laboratory of Patrick… Continue Reading