Malaria

7. What was the most significant finding of the microarray experiment? Why is it significant?

**Microarray is a technique to look for gene expression (nothing to do with size or function of genes so choices B and D are not relevant here). Most genes in the genome that are expressed at decently high levels, throughout the asexual lifecycle, exhibit periodic expression: they are induced and repressed approx only once per cycle "The genes are expressed only once in Plasmodium life cycle, then turn off" ******This finding is significant because, since plasmodium's gene expression is very stage specific and their development occurs in a stepwise function, researchers think that if they can make drugs that will disrupt gene expression at one stage, it can derail the parasite's development.

6. What is DNA microarray? What does this technique allows you to do?

**Microarray is a technique to look for gene expression (nothing to do with size or function of genes) **Microarrays are used for anytime when you want to compare expressions of ALL THE GENES IN THE GENOME OF AN ORGANISM Ex: What are the genes that cancer cells express vs healthy cells DNA microarray approach helped them to find more about what genes are used and when throughout the asexual blood phase of the parasite.

The Blood Stage: asexual cycle of the parasite

**don't need to remember the names of different forms of parasite **The main point is that the goal was to find out which genes are expressed at which stage. They did this by doing a microarray experiment => sporozoites injected by mosquito => merozoite is the form that bursts forth from the liver and goes on to infect erythrocytes => after invasion, the parasite called a ring stage because it resembles a small ring on the inside of the erythrocyte => then the main metabolic phase of the parasite falls, called the tropoziote phase => then parasite goes through multiple forms of replication to ultimately form what's called a schizont, which will then mature, burst again, and release merozoites to repeat the cycle

4. What stage of the parasite causes most of the symptoms? What happens during this stage to the host?

**don't need to remember the names of different forms of parasite 1) Infected female anopheles mosquito bite 2) releases sporozoites from her salivary glands into the blood stream 3) sporozoites travel to liver to invade hepatocytes (liver cells) where they reproduce into merozoites ***ASYMPTOMATIC 4) "The asexual erythrocytic cell cycle of the Plasmodium parasite" aka "The blood state" (it is the parasites lifecycle in a red blood cell - see next card) => Merozoites burst from infected hepatocytes and invade other red blood cells, consuming the hemoglobin as food source ***this is where all the clinical manifestations of the disease occurs and where most drugs attempts to eradicate the parasite ***this results in an exponential increase in number of parasites and more and more red blood cells become infected 5) some small number of parasites develop into a sexual form called gametocytes 6) these gametocytes can be picked up again by another mosquito, undergo a development in there, then start the entire cycle again

Two approaches to antimalarial drug development DeRisi lab approaches to finding new drugs:

1) Priviliged Scaffold: => Start with chloroquine chemical structure and modify it 2) Diversity Screen: => Probing for antimalarial activities amongst known drugs => Benefit with this approach is that it is a short cut from the privileged scaffold approach because you are looking at drugs that have already passed toxicity testing, etc **In both cases, the researchers test the effectiveness of the new compounds in disabling Plasmodium's ability to infect red blood cells (RBCs). They measure this using a flow cytometer. **Using priviliged scaffold approach, the compounds need to be tested to make sure it is safe. But with diversity screening approach, this testing has already been done.

2. What pathogen causes Malaria? How is the disease transmitted? Which cells are most affected?

=> Caused by one of four parasitic protozoa **Plasmodium falciparum is the most deadliest of the four parasites and has largest distribution worldwide => Transmitted by Anopheles mosquito (over 60 species of this mosquito that are capable of transmitting the disease) => hepatic cells and erythrocytes are most affected

How to weed out the bad vs the good compounds in the screening

=> Solubility => Genotoxicity => Cytochrome p450 inhibition - tests whether these drugs will interfere with the metabolism of other drugs => Permeability - a proxy of measuring bioavailability => How well the drug partitions into erythrocytes relative to blood serum => Human Liver Microsomal stability - how stable is it against liver enzymes? **First have to go through all these stages and only then can you go on to test in organisms => Rat Oral Pharmacokinetics - how well does it get into the blood, is it orally available, does it have any toxicity against other organs, kidneys, etc Additional demands from a new drug *Needs to be cheap (~10 cents/pill) *Must be hard to develop resistance to *Must have minimal side effects *Must be able to be used in combination with other anti-malarial drugs

CQ: What are the major symptoms of Malaria?

=> periodic febrile episodes (fever) => chills, sweats, aches, nausea, weakness => enlargement of spleen Worst form: => seizures, coma => severe anemia => organ failure **Most of the deaths occur in Africa, mainly in children < 5 years of age

Developing new drugs: always need an assay that will allow you to tell if a drug is effective

A 72 hours whole cell growth essay measured by flow cytometry. They treat the parasites in the ring phase. They let them go through two cycles and measure the increase in the number of infected cells, over this 72 hours (each cycle is 48 hours). How do we measure the number of infected cells?

Why is there a high frequency of Sickle cell anemia alleles and Thalassemia in Malaria rich regions?

Because the parasite has trouble infecting those sickle cells. This gives heterozygotes advantage against malaria and malaria exerts a selective pressure for them.

What is the most frequent method for diagnosing an infection by plasmodia?

Blood spear. Take sample of blood and stain with a DNA stain. Healthy blood cells don't have a nucleus or DNA but infected blood cells has the parasite DNA.

CQ: Red blood cells were incubated with a fluorescent dye (FITC-A) that binds to DNA. In the flow cytometer image on the right, the X axis is the amount of fluorescence, Y axis is the number of cells. Which sec=on represents the infected cells?

C

Parasitemia

Cytometry will allow us to measure the parasitemia: the percentage of cells that are affected. Flow cytometer: an automated fluorescent microscope, cells pass the objective very quickly, ~30,000 cells per second. Fluorescent dye stains DNA, only infected erythrocytes will fluoresce (red blood cells have no nuclei). **X axis = amount of fluorescense **Y axis = number of cells **The graph to the left is what we are trying to achieve.

(2/2) How does Chloroquine and other quinoline antimalarial drugs work? A mechanism of action:

How does quinoline antimalarial drugs hault hemozoin formation? => All these Quinine-like drugs work by binding to the elongation site of the heme polymer (hemozoin) and preventing further additions of heme. How does quinoline antimalarial drugs kill the parasites? => These FP-chlorquine complexes are free and not crystallized. The complex is highly toxic to plasmodium, disrupting its membrane function and resulting in plasmodium cell lysis.

In 1950s it was thought that DDT and chloroquine could enable us to eradicate Malaria. What happened?

In 1960s resistance toward DDT in mosquitos and resistance to chloroquine in plasmodium started developing

3. Why is the disease still such a serious threat? Be able to address: current medications (no need to remember the names, address why are they not effective), vaccines, public health efforts.

Limited acquired immunity requires repeat infections WHO Global Eradication Effort: 1955-1987 failed because => drug (Cholorquine) resistance => insecticide (DDT) resistance => bypassed Africa => lack of funding => lack of community participation => war and population movements

How does a pathogen develop resistance?

One way There are selective pressures on the pathogen population to develop mutations that will provide it resistance. Another way has to do with patients and their usage of drugs. If low concentrations of the drug is in your system, not enough to kill all the pathogens but just enough to apply a selective pressure to develop mutations that would incur resistance.

A 72 hours whole cell growth essay measured by flow cytometry

Parasitized red blood cells have DNA. Staining those cells with fluorescent dye will allow us to count them with a flow cytometer and differentiate those from noninfected cells.

Malaria: the pathogen and the vector

Pathogen: single cell parasite from the genus Plasmodium Vector: mosquitoes (genus Anopheles) **infected females

(1/2) How does Chloroquine and other quinoline antimalarial drugs work? The weakness of plasmodium that they exploit:

Plasmodium feeds on hemoglobin. However, FP (in the Heme of hemoglobin) is highly toxic to the parasite. To avoid toxicity, the parasite biocrystallizes heme into non-toxic hemozoin crystals.

5. Why so few drugs have been developed?

Quinine => quinine resistance => quinine supplies were under control of imperial Japan Chloroquine => chloroquine resistance Mefloquine (aka malarin) => mefloquine resistance Halofantrine => cardiotoxicity in certain individuals so cannot be considered as a drug for worldwide use Artemisinin => ***very few compounds have been developed every year/decade for malaria bc its endemic in the poorest part of the world and there is not a large profit incentive for major pharmaceuticals to develop this ***The days of mono-therapy are over. It is not possible to rely on a single drug to help eradicate it. To bypass resistance or to lower probability of resistance occurring in new parasites, we must use drugs in combinations.

Problems with DDT and chloroquine

Resistance toward DDT in mosquitos Resistance to chloroquine in plasmodium

The Mutagenic Chain Reaction (MCR): Chromosomal Insertion into one allele, CRISPR step 1

Step 1: => Using CRISPR/Cas9 with a gRNA against noncoding region of the mosquito genome => Inserting a plasmid containing a construct with Cas9 and gRNA between the homology arms => Using the plasmid as the template for homology directed repair for a chromosomal insertion into one allele

MCR conversion of sister allele, CRISPR step 2

Step 2: => Now the Cas9 and gRNA is expressed from the modified allele on the genomic DNA of the mosquito => Now the gRNA guides Cas9 to the same exact location but on the other chromosome => HDR is using the modified allele on the other chromosome as the template

Up until now we were talking about drugs that target plasmodium. The following approach targets the vector itself (the mosquito)

The gene AgCP was discovered. This gene is required to for mosquito to process the blood meal. The promotor of this gene is turned on by components of a blood meal. Researchers took this AgCP promotor (that turns on with blood) and put it behind a gene that codes for SM1 single chain antibody, which binds to SM1 in plasmodium and blocks it from infecting humans.

How do we deliver this transgene to the entire population of mosquitos?

The original idea was to breed huge quantities of these transgenic mosquitos and release them into the population. These homozygous males would mate with females and all their progeny will be heterozygous - which is okay because it's enough to have just one copy of the transgene for this method to work. However, as the population breeds, these allele will get diluted, requiring you to resupply more transgenic mosquitos. Here is the discovery: UCSD Researchers used a modified CRISPR/Cas9 system to create an allele that spreads and multiples

Where can you target malaria in its life cycle?

Vector control: taking out the mosquito (if you have the money the resources, etc) Vaccines: seek to try to block the initial infection inside the liver (so far no vaccine with an operational impact) Chemotherapetic Drugs: target asexual lifecycle (entererythetic? life cycle)

How does DNA microarrays work?

What is a microarray chip? => A solid surface with a collection of DNA spots attached all across the surface. It has the whole genome spread out across the surface and each gene is at a known location. How it works? => Extract mRNA from two different stages => Reverse transcribe into cDNA and label the cDNA from each of the stages with a different dye => Then hybridize to a microarray (a chip with all of the genes stamped in known positions) Example of interpreting results => Say you see hybridization from the red cDNA to a particular spot on the microarray chip. We know gene x corresponds to that spot. So the cell expressed the mRNA for gene x at the time the mRNA was extracted => Say you see hybridization from red cDNA and green cDNA (from another sample where the cell was in a different stage) both at gene x. Then we know gene x is expressed at both stages. Why do we first convert mRNA to cDNA? Why not just hybridize the mRNA straight to the microarray? => Because cDNA is more stable

The development of Artemisinin

Youyou Tu identified the active ingredient in an old anti-malarial remedy, the wormwood plant Artemisia annua Isolated it as a chemically pure compound: Artemisinin Here's the story: She extracted from the plant mentioned in ancient text a chemical that inhibited malaria mice but it's activity was inconsistent She turned back to the ancient scripture and read about extraction from the leaves using cold water. She used a cold extraction procedure she developed that uses ether rather than a previously used ethanol extraction. Temperature extraction allowed Tu and her team to extract the compound that consistently killed 100% of malaria parasites. Artemisinin structure and mechanism is different from the quinoline antimalarial drugs

Therapeutic Index

the concentration that they kill malaria at, was similar to the concentration that they're used at for normal clinical purposes.

Summary: Using MCR to eradicate malaria

• The idea is to generate transgenic mosquitos that express the antibody (scAb SM1) that prevents mosquitos from transmitting malaria. MCR approach allows the transgene to spread through the mosquito population. • The transgene includes a promoter that is turned on by blood meal. This promoter controls the expression of a single chain antibody (scAb SM1) that blocks the infectivity of the mosquitos. • This transgene also has Cas9 and the guide RNA against a region in the mosquito's genome that is neutral to its survival. All three are surrounded by Homology Arms (HA) - regions identical to the neutral region in mosquito DNA. • CRISPR/Cas9 system facilitates the integration of this construct into mosquito genome, using homology recombination DNA repair. • The beauty of MCR is that once one copy of the transgene enters one chromosome of the mosquito (let's say, mom's chromosome), the same system converts the other allele (dad's) into antibody gene-bearing allele as well. So if a homozygous transgenic male mosquito mates with a wild type female, all of their progeny (and all of their progeny's progeny, etc) will become homozygous for the antibody gene as well. Thus, this approach can offer a way of converting a population of mosquitos into non-infectious in terms of malaria. • Other diseases could be targeted with this system as well...

MCR conversion of sister allele: now both alleles contain CRISPR/Cas9

The result is now both alleles have the construct

How DNA microarrays can be used to make "expression profiles"?

Allows you to see when every single gene in the genome is expressed. With this information, you can make an expression profile. **They saw that plasmodium genes are expressed synchronously (related to next slide) *In contrast, with humans, if you were to take all the mRNA from all our cells at any given time, you'll see most of our genes on because they are expressed at different types of cells. Our cells are not synchronous where they do one thing at a time.

Medelian inheritance vs MCR inheritence

Medelian inheritance: => 50% allele inheritance => 100% wild type MCR inheritance: => 100% allele inheritance => 100% mutant

CQ: Which of the following is NOT the reason for lack of effective treatments to Malaria?

B Problems with following treatments: **The indoor sprays with insecticide DDT has been used less due to the concerns of leakage to environment (environmental concerns). **Bed nets sprayed with insecticide are effective but re-application of insecticide is needed and this can be a problem.

CQ: Which cells are most affected?

D