WILLIAM PALMER: Good afternoon, everyone. My name is Dr. William Palmer, and I'm Associate Director of Education for the Center for Individualized Medicine in Florida. I'd like to welcome you to the Florida Center for Individualized Medicine Grand Rounds series. The CIM Grand Rounds lecture series is designed to highlight the latest in scientific discovery and innovation, and to demonstrate how individualized medicine is being translated into our practice to meet the current and future needs of our patients.

It's my pleasure today to introduce Dr. Ravi Durvasula. Dr. Durvasula is the Chair of the Division of Infectious Disease within the Department of Internal Medicine at Mayo Clinic in Florida. He is consultant and researcher who aims to reduce the burden of parasitic and viral diseases in the global population by addressing both insect-mediated transmission and drug therapies for humans. His focus is developing new therapeutics and strategies to control transmission of infectious disease.

As a Howard Hughes post-doc fellow at Yale, he developed a model for expression of antitrypsin molecules in the gut of a Chagas disease vector. This work was funded by the NIH, and resulted in several additional lines of research into infectious diseases that involve strategies for design, expression, and characterization of recombinant molecules that can be used for control of infectious disease transmission.

He's pioneered new classes of intrinsic fluorescent antibodies and new strategies for in-vitro ribosomal display, and he holds several patents in these areas. Over the past few years, his lab has expanded its work into the field of molecular design and drug discovery, and several programs with international collaborators are underway to design and develop new therapies for parasitic diseases, such as malaria and leishmaniasis, and illnesses caused by emerging viruses, such as SARS-CoV-2, the Zika virus, and Ebola. His molecular design laboratory is developing new antibody-based therapies for non-infectious conditions, such as chronic pain and post-ischemic stroke.

Today's presentation is a drug discovery platform for novel therapeutics against leishmaniasis and malaria. He will cover a comprehensive exploration of new drug discovery platforms designed to tackle leishmaniasis and malaria. This discussion will begin with an overview of the global epidemiology of leishmaniasis infections, setting the stage for a detailed look at various clinical syndromes associated with these infections and the limitations of current therapeutic options.

We'll then delve into some advanced molecular approaches employed to control leishmaniasis. And additionally, the presentation will address urgent need for novel therapeutics against malaria, and highlight the ongoing drug discovery efforts at Mayo Clinic aimed at addressing these clinical health challenges. So we're going to get started here, and I will turn it over to Ravi.

RAVI DURVASULA: Thank you very much. Let me share the screen. Perfect. OK. Thank you very much, Dr. Palmer, for the introduction and the opportunity to address this very large group, and certainly talk about some-- I also want to thank by the way, Ana Muskaj for her help in helping put all this together.

I will be talking about two diseases, primarily leishmaniasis and also malaria, things that may not come up as often as I'd like in some of these kinds of venues, but are very important on a global stage. And as Dr. Palmer said, I work here in Florida as the Chair of Infectious Diseases, and have worked for many years now both in Clinical Infectious Disease, as well as running a basic science molecular lab, looking at some of these very important parasitic illnesses. So let me get started and see here. There we go.

So in today's seminar, the objectives are to review the global epidemiology of Leishmania infections and to try to provide the summary of some of these clinical syndromes so everyone can recognize what these are, some of the current therapeutic options. And to really set the stage or frame the concept of newer and evolving therapeutics for some of these diseases, and the mandate for these kinds of therapeutics, many of which are highly, highly individualized and designed around some of the molecular modeling.

And then to also include the mandates for malaria and where we are currently with the global malaria picture. And to spend the last part of the talk on some of the drug discovery efforts going on here in Florida with our international collaborators, and show you, actually, some of the data from the lab on newer therapeutic options.

I'm going to start with an overview of leishmaniasis. And this is what's called a vector-borne disease, a disease that is transmitted by arthropod vectors. Shown here are the sandflies. And like a lot of these diseases-- and certainly malaria will be in the same category-- we have to think about factors that are related to the parasite, the pathogen, in this case, parasites of the genus Leishmania; the vectors, which are sandflies, and I'll go into a little bit more on each of these; and then what happens when these come together and hit the human host and various clinical manifestations, which I will walk through.

Here's an important theme when we look at not just Leishmania infections, but also malaria, when we look at a variety of global pathogens. And that, by the way, is something that's a major focus of our Infectious Diseases division here in Florida, is to look at global and emerging pathogens, not only through our own laboratory-based investigations, but also with partners around the world, and I'll show you some pictures of places where we work.

But Leishmania and the Leishmania syndromes-- and really, we talk about this, as shown here, as leishmaniasis. More correctly, it's the leishmaniases, with an E there, because there's a whole spectrum of diseases. And what's shown on this map are really three of the cardinal features of this disease complex. What's in red are the cutaneous forms of this disease, and I'll be showing you some pictures of these in a minute.

But that has a true global distribution. Hundreds of millions of people are in at-risk areas for these sandfly-mediated cutaneous diseases. About 12 to 15 million are actually infected. Now, I want to put a little caveat on this, that when we talk about some of these global diseases-- and especially those that disproportionately affect people of lower socioeconomic strata and diseases of poverty, what we call neglected tropical diseases-- epidemiology is very crude. So when I give numbers here, please understand that the numbers could be much, much larger, and probably are, because reporting and surveillance can be quite spotty in many parts of the world.

But the cutaneous form is truly distributed globally across all continents, except probably Antarctica. The visceral form of this disease, shown here in the kind of greenish coloration-- again, I'll give you more details-- is much more geographically restricted. And then there is a form, known as the mucocutaneous, shown here in the purple, predominantly in Central and the northern parts of South America.

This is a disease, like a lot, where the epidemiology is rapidly changing. And if I were to just focus here on the Middle East and the areas around the Arabian Peninsula, Israel, Syria, etc, there's been an absolute explosion of cutaneous disease, largely triggered by societal unrest and all the things that we know have happened in the last 10 to 15 years in this region.

So case identification in Syria and Afghanistan and Israel, places like this, has really gone through the roof, as well as changing patterns of disease, which we'll talk about in a minute. And this, again, is an important theme when we talk about diseases of global significance, how closely tied they are to the state of society, functioning in a society and unrest.

This is drilling down into the visceral form of the disease. And I put this up here as a global map to highlight, once again, that the traditional areas, hotspots for this disease, have been in parts of East Africa, the very northeast of India, into Bangladesh and Nepal, and some pockets in Brazil. But if you notice on this map, you see kind of a ring here around the Mediterranean, including parts of south Europe, and an area where I've done a lot of work on this disease, in Tunisia, where there's been some increase. And this reflects the overlap of this disease with HIV/AIDS and the reporting of visceral leishmaniasis and people that are co-infected. Again, an example of how changing patterns of disease reflect on the global epidemiology and the impact of it.

So my lab, for many years-- I'm a true biology geek-- we have actually maintained populations of vectors. I've done a lot of work on the vector biology of these infections. So when we talk about Leishmania, we are, by definition, referring to sandflies. These little critters-- by the way, all over Florida you can find them. They're ubiquitously distributed, small flies that suck the blood from humans. I'm going to show you the life cycle in a minute.

And we generally refer to two broad categories, those of the genus Lutzomyia, which caused the New World or the American leishmaniasis, and then those of the genus Phlebotomus, which are the Old World. I'm not going to focus a whole lot today on the vector biology, though I will show you, a little later, just a reference to some of the work that has been done in my lab with these.

The parasite-- and this is a Protozoan parasite. I'm going to give you the classification in a minute. But Leishmania-- and I know there probably are no medical students on this talk, but this is, as you know, from your days in medical school, how we torture medical students, is to remember things like this. And this looks, at a glance, as a very, very busy, complex slide. But I put it up here because I want you to appreciate that we are talking about a very broad class of diseases, the leishmaniases, divided by geography, where they're located; divided by the form of disease-- cutaneous, visceral, or mucocutaneous; by the parasites; and the vectors.

So it is a complex classification, but in general, kind of some themes for people to remember. The mucocutaneous version, shown at the bottom, are, by and large, in Central America and the Amazonian regions of South America. So that's the northern part of that continent. The other forms of this disease, the cutaneous and visceral, can be seen in different parts of the world.

If we look at the visceral, there are forms of this, which are largely what we call the Old World forms, of South Asia, a little bit now in the Middle East regions, and then Africa, in the eastern part of Africa. There is a visceral pocket, as well, which I showed you earlier, in Brazil and some of the northern regions of South America. And then cutaneous is ubiquitously distributed, as I mentioned, throughout the world. So it's quite a complex categorization, as I say, with different parasites, different types of infections, vectors, but a fascinating global distribution.

And if we look at this-- and here's the other part of, again, the PTSD of medical school-- remembering parasite life cycles. And this is courtesy of the CDC. I put this up here because it's going to be important when we talk about drug targeting and where the focus is. Vector-borne diseases, by definition, there's a vector phase. There's the parasite's life cycle in the arthropod, in this case, the sandflies. And then there's the human phase, face shown on the right.

And it's the transmission from the sandfly to the human of what are called promastigotes that get internalized by macrophages. And this is a very important theme when we talk about drug discovery, and that is the intracellular life cycle of the Leishmania amastigote, which is now what's inside the macrophage, and its ability to survive, which becomes the focus of drug targeting. So as these multiply and the macrophages are then acquired through another blood meal-- by the way, these are only female sandflies that do all this. That goes back into the insect host. And then you get replication into these promastigotes in the gut of the sandfly.

Notice here-- and you're going to contrast this with malaria, the Plasmodium life cycle-- this is purely binary fission. There is no sexual phase of reproduction of Leishmania. Therefore, the genetic diversity of this parasite is far less than the malaria parasite. But that is your general cycle of leishmaniasis, the parasites. If

We look at the clinical spectrum-- I already alluded to this-- three broad categories. The one that is globally distributed, the cutaneous. And this here, this picture, is the classic cutaneous ulcer of leishmaniasis. It's an ulcerative lesion with what they call heaped-up pearly borders. And these can be painful, initially, but they can also be quite chronic and painless.

Often-- not often, almost always-- in areas that are exposed. So you see these on the forearm. You can see them on the legs, where the skin is exposed, and it causes this kind of a lesion. The extreme other end, what's shown in this unfortunate, young fellow, is the visceral form of the disease. And here you see somebody-- this is a younger child-- with visceral leishmaniasis. And all of that, what you're seeing in the belly, is spleen and liver, massive hepatosplenomegaly.

And in between, in those geographic regions I mentioned of Central and the northern parts of South America, is a disease called Asplundia or mucocutaneous leishmaniasis, which is really an invasion of the mucous membranes of the nose, the mouth. Generally very disfiguring. Not fatal. The visceral form is universally fatal if not treated. The cutaneous form is rarely, if ever, fatal. This can be, of course, if there's airway collapse. So it depends on how it is. That's your general spectrum of leishmaniasis.

Now, in terms of the cutaneous form, the isolated version, what I just showed you in the previous slide here, can often be watched. In a healthy person-- I've seen many of these cases in travelers who return from doing fieldwork in Central America or Asia-- a lot of times these can be watched. Maybe topical care to keep it from being infected. Super infected, I should say. And then it will go away.

In the disseminated cutaneous leishmaniasis-- and this is where it starts to get a little bit more complicated-- where multiple-- so this is someone's arm-- multiple, multiple lesions are occurring here on the arm. And these can be very disfiguring. These can also cause bacterial superinfection. We're also seeing-- and this is part of the changing epidemiology-- increased disseminated leishmaniasis in that area around the Mediterranean, also in the setting of HIV and immunosuppression.

So in these cases, systemic therapy with things like amphotericin B, miltefosine, and pentavalent antimonials can be used. I'm going to talk about these a little bit more with the visceral form. But one can use systemic therapy here. And there's also been a lot of good work done, especially in northern Africa and the Middle East, on cutaneous disease being treated with heat. That's photodynamic and topical heat therapy with some very, very good results. But this can lead to disfigurement and morbidity.

And interestingly, there's a whole historical side to Leishmania, Leishmania infections, where when we talk about those isolated lesions, like this, in many of the Old World societies, the goal for many societies was to prevent, especially young ladies, from getting these kind of lesions on their face, because of disfigurement. So there's a practice in many societies of taking young girls and having them bitten by sandflies in areas of the back and the lower regions in the trunk, that aren't exposed, as almost a form of immunization, to prevent subsequent lesions in the face. That, of course, is the basis for some of the new work that's being done with Leishmania vaccines.

So that's the spectrum of cutaneous disease. This is the deadly form. So 50,000 to 75,000 people a year die of this disease, called visceral leishmaniasis. And this is when the disease runs amok, from just being cutaneous to invading the reticuloendothelial system. What's shown here, this is a picture of a patient that I had a chance to meet and treat in Bihar, India. And again, what's outlined is the liver and the spleen. Massive hepatosplenomegaly.

These patients who develop visceral leishmaniasis often go on to develop severe immunosuppression with a drop in globulins, gamma globulins drop, and then secondary bacterial infection, because of both the immunoglobulin deficits, as well as the splenic dysfunction that occurs in these patients. So a lot of times we see these patients getting disseminated bacterial sepsis and death. And this is, again, an inexorably fatal disease if untreated, and it can be quite devastating. Worse outcomes in HIV patients.

I showed you, previously, some new pockets of this disease. A place where I've done a lot of work is in Bihar, India, where they have a center that has the world's largest co-infection, cohort of people with HIV, what's called kala-azar, the visceral leishmaniasis of South Asia, and cases and manifestations that will not be seen anywhere else in the world.

The treatments. I want to take a moment to talk about these because this sets the stage for the mandates for new therapeutics. Liposomal amphoB works beautifully. Up to very high, high doses are given, but it's curative in these patients. Keep in mind the cost of this drug. Even in the US, this can be very expensive. When you get to parts of the world where these drugs are extremely hard to get, you understand where some of the problems come up.

Miltefosine-- and I'm going to show you where it works on the calcium cascade-- can also be used for up to 28 days. And this is used primarily for L donovani, which is the South Asian variant of visceral leishmaniasis, the kala-azar variant, they call it. Not used for women who are pregnant or breastfeeding. Can be very, very effective. Also, price can be a concern with that. And the age-old treatment, pentavalent antimonials for 28 days. These are injected into a person.

And I call this the form of torture. It's painful. It can dramatically decrease the renal function, so a high rate of renal toxicity. And now very, very prevalent in some areas. More than 50% rates of resistance of the target parasites. So pentavalent antimonials may be cheap. They've been around. But they're toxic and they don't work very well.

So when we look at Leishmania as a group and the different syndromes, we see-- and this is really where I want to move with this-- the mandates to move forward with new therapeutics. One, vector eradication. There has been a lot of work in India, as I mentioned, in the state of Bihar. DDT spraying has been done rapidly to kill sandflies. And there are parts of Bihar where the DDT is actually now a defoliant. It wipes out foliage from trees, and the sandflies are unscathed.

These are resistant to that pesticide. So resistance is an issue. It costs a lot of money to do this. Sustainability of campaigns like this become very difficult. I mentioned the cost of some of the drugs, like liposomal amphotericin B, and getting it to people who are affected, who are, again, a very low socioeconomic group primarily. The toxicity, the evolution of parasite resistance-- all of the classic red flags and pitfalls of dealing with a neglected tropical disease.

My lab, as I mentioned to you-- and I'm not going to spend much time on this, but it might be of interest-- we have worked for years on strategies called paratransgenic approaches to vector-bone diseases. And the basic premise of this is that if you can't beat them, can you at least modulate them? We can't eradicate vectors of disease. Wiping the earth clean of insects is an impossibility. We'll kill ourselves before we kill the insects. But can we change them? Can we modulate their ability?

So our lab has done a lot of work on identifying endogenous bacteria of these phlebotomine sandflies, and engineering bacteria to produce molecules in the fly that block that cycle of the parasite. Remember, I showed you the vector side of the parasite. And this is from a previous publication. We're using spectral imaging. We've actually had a sandfly here, which is now expressing green fluorescent protein through its endogenous bacteria, and the sandfly actually glows. If you will, a sandfly that's become like a firefly. So the concept is to express these molecules. And of course, there's a whole slew of them that are designed to target Leishmania as well. That's another topic, but something that I thought you might be interested in.

Let me say a few words now about malaria. And this is one that, obviously, people have heard much more about. Another vector-bone disease, and you're going to see many parallels to the leishmaniases. Here, the vector is this, the Anopheles mosquito. Once again, the female Anopheles mosquito, which is the one that transmits the malaria parasite.

If we look at it, it, too, has a global distribution. I always tell the med students that malaria is the prototypic tropical disease. Because if you took this globe, this map, and drew the two tropics on here, you will find that the bulk of malaria cases fall right in between. So this is the classic tropical disease, and again, with the global distribution that has been like this for a good long time.

When we look at the numbers, this is the most deadly of the parasitic diseases in the world. And it's a shame because this is a disease that is treatable and preventable. If you look at these numbers of nearly 660,000 people dying each year, these numbers used to be much higher. So this is an improvement.

But notice, 200,000 newborns. Maternal malaria, which is deadly. Childhood anemia. Cerebral malaria. Many, many manifestations of this disease that are deadly. This is a much more virulent parasite, especially the falciparum form of this disease. These numbers I mentioned are improved. They used to be worse, 30%, 40% higher. And concerted efforts, with things like bed nets-- the female Anopheles mosquito only bites at night, dusk to dawn, so sleeping under a bed net is the most important way to prevent malaria. There is no-- as you know, the vaccine still remains the Holy Grail and has not been deployed yet.

But we made a lot of progress globally with the control of malaria. And then with the COVID pandemic, many things fell behind. And we all know that a lot of the infrastructure of health care and prevention fell behind around the world. Unfortunately, cases of malaria have actually risen again. So hopefully, we will see that improve.

But it remains an extremely important disease. Here's another one of these maddening life cycles. But I put this up here to point out that, again, the insect, or arthropod stage, the human stage. And notice that as the mosquito bites and injects what are called sporozoites into the human, the first target is actually the liver. So the hepatocyte gets infected and one gets these hepatic stage parasites, which form a schizont. These then rupture into the merozoites.

And it's this cycle, in the red blood cell, the erythrocytic stage, that we think of as the classic malarial syndrome. Intermittent fevers. People sometimes use the periodicity of the fever as an index of the type of parasite, which malarial parasite. There are four classic falciparum malaria and vivax, but new ones have been discovered-- Plasmodium knowlesi, which is in the Malaysia, Singapore area, and Plasmodium brasiliensis, which is now being seen in the Amazon regions.

But we think about this cycle here. Note, once again, the intracellular nature of this infection. Very, very important as we talk about drug discovery. The other important feature of malaria, which distinguishes it from the Leishmania cycle, is right here. And that is you now have a sexual stage for that parasite's life cycle.

As they go through this cycle of intraerythrocytic maturation, you also get gametogenesis, so gametocytes form. And when those fuse and enter back into the mosquito, the female mosquito, you have an entire sexual phase with the development of ookinetes, oocysts, and then the whole thing begins again with sporozoites that go through to the human.

And this here explains it all. It is that added step, with the genetic diversity, that leads to huge amounts of antigenic variation, immune evasion, and many, many of the issues that we face with things like vaccine development. That's the malaria life cycle. And we always think about-- as I say, there's a beautiful picture from the CDC and EM, micrograph of infected, or these are parasitized, red blood cells, showing the rupturing here as these parasites are leaving. And of course, from the clinical standpoint, this is what we find when we do a smear of a patient. This is falciparum malaria with the ring forms of the parasites, seen intracellularly, and then many membrane associated changes that occur.

So as we talk about malaria-- and again, I'm not going to get into too much on therapeutics here-- a few of the same themes still apply. Here at Mayo Clinic, we see many, many patients who are traveling who are given medicines to protect against malaria. And some of you who have either traveled or practiced in this field know about atovaquone, proguanil, or Malarone. That's the one we use the most. Sometimes we use other drugs that are given weekly, and Primaquine for that terminal prophylaxis, which gets at the hepatic stage, where you can have dormant or intracellular forms.

And then the treatment choices here, again, uncomplicated malaria. Relatively straightforward to treat here. The complicated forms, we use artemisinin as the backbone in a combination therapy. IV artesunate, which is used, and I've used it several times, comes from the CDC on an as-needed basis. So these remain the backbone, but just like I mentioned with Leishmania, the emergence and expansion of the artemisinin-resistant P falciparum has been well-documented. This is a paper describing what's been going on in Rwanda.

There have been isolates from Southeast Asia, the border of Cambodia, Thailand. There have been isolates from Northeast India as well. And the thought of drug-resistant P falciparum malaria in a highly malarious country, like India, is chilling. So once again, emphasizes the changing nature of this infection. New parasites are being discovered. Resistant parasites are being discovered. And there's an entire subject, another seminar, entire body of work on the vector side of this, changing migrations, changes with global climate change, etc. Many, many moving parts when we talk about malaria.

I put this up here because we're in Florida, and so I have to, have to talk about this. Last year, the CDC and the MMWR reported seven autochthonous cases from Florida and one from Texas. So an autochthonous case is a case that's not imported. So this is malaria transmitted by Florida-grown, Florida mosquitoes, to Floridians and then the one in Texas.

This is scary. So this means that this disease has actually indigenized in this country. If you think back many years to the West Nile virus invasion in and around New York City, again, this is the kind of concerns that we have. These cases were all due to Plasmodium vivax, so not the most deadly form, which is falciparum.

All were treated. I mentioned to you the simple three day treatment courses. So all were successfully treated. But interestingly, in Florida, all the patients, seven of them were within a four-mile radius in Sarasota County, and no travel or relationship to imported cases. So needless to say, the state of Florida has blasted with pesticides to try to control this, much like what was done 20 plus years ago when West Nile virus hit New York and Connecticut. So something to keep in mind. And again, the concept of emergence, reemergence, and the changing nature of these diseases.

So to put all this together, the challenges. These are diseases of enormous global public health impact. Hundreds of millions of people at risk, tens of millions of people infected. High mortality, especially with malaria. Changing patterns of disease. Changing patterns of leishmaniasis. Changing patterns of malaria. Vector migration. Vector resistance. Emergence in new areas and new forms of it.

The sustainability for things like vector control. How sustainable is it to keep trying to eradicate these? The evolution of parasite resistance, something which has greatly hampered Leishmania and is now emerging in a way that it could knock out the very backbone of malaria treatment, which is artemisinin.

The lack of vaccines. Colleagues of mine and my own lab have been involved with a group that's developing centrin knockout parasites for vaccine development against Leishmania, and there are now trials being done in several parts of Africa and Asia on these vaccines. But still, nothing that is ready for prime time. The malaria vaccine has received billions of dollars of support from the NIH and DOD and many, many other groups. Still, I think, on the horizon, but not there yet.

So we don't have the vaccines. Many of these therapeutics are expensive. And when you consider who's affected disproportionately by these diseases, these are diseases of poverty. They're neglected. So all of this comes together to create these global challenges. And really, it is an issue of health equity across the world when so many of the people who succumb to these diseases are poor. So that brings me to where we are headed. And hopefully at this stage, you can appreciate the importance of the global challenges.

Now, if you go back to those two life cycle slides-- and I apologize upfront. This is a busy slide, but I'm going to walk you through it carefully. The next few slides are slides from the lab. So you're going to see some molecules and computer simulations and things of this sort. Both the parasites of Leishmania, shown in this cartoon, and the Plasmodium parasites that cause malaria depend on intracellular homeostasis. If they cannot survive inside cells, the parasites die, and that's it. So it's a very important piece of the life cycle of these parasites.

And obviously, maintenance of an intracellular life cycle for any organism-- whether it's these parasites, whether it's bacteria, like listeria, etc -- there are many, many complex adjustments. But one of the most important ones in these parasites is the ability to regulate calcium. So calcium is something that is required in the life cycle of these parasites at many, many stages. Again, another 10 seminars on calcium biology and parasites.

But everything from entry and attachment to intracellular cycling and functioning to intracellular maintenance and immune evasion to maturation and differentiation of the parasite, even at the vector stage-- all of these are critically dependent on calcium. That shall be the focus as we go forward, and you hopefully can appreciate this. So what's shown in the top here, the sporozoites of Plasmodium falciparum-- I mentioned to you the first target, the hepatocyte, the liver cell.

Forming the merozoites. Release. Rupture. Forming the trophozoites inside the red cell. So this is now the red cell, which then goes into two forms, the schizonts, which rupture and the whole thing starts again, or the gametocytes, which then go on to form the new ookinetes. This is what I showed previously.

Right in here-- and again, I apologize in advance for all of these. We had a grad student who just went nuts with this slide. But what I want to do is focus right there on artemisinin. I'm trying to circle it right here, artemisinin. The backbone of therapeutics for P. falciparum, targeting something called the SERCA. This is the endoplasmic reticulum calcium storage mechanism.

And this is analogous to what's also found in the sarcoplasm in human and vertebral myocytes, and is associated with sequestration of calcium. So that ability to sequester and store the calcium is critical. Artemisinin disrupts that. So it's a calcium-acting drug, which is used to kill the parasite in its erythrocytic stage. Some of these others are still experimental, so I'm not focusing on them.

When you look down here, this is the Leishmania life cycle. And again, I mentioned to you sandfly bite, promastigotes. And the promastigotes very quickly enter the macrophage through phagocytosis. So unlike the liver cell and the red cell, this is all inside the macrophage. And inside the macrophage, you get development of what's called the amastigote, and that's taken back in by the sandfly. The whole thing repeats without the sexual stage.

In here-- again, multiple experimental drugs-- but right down here, miltefosine. Remember I mentioned, this is one that's used in South Asian leishmaniasis, kala-azar, and one that targets the acidocalcisomes. Here's another intracellular organelle, which is critical to the survival of that Leishmania parasite. And I'll show you some beautiful video later on of where our drug targets work.

And the acidocalcisomes is actually the area where, as the name implies, it's a highly acidic organelle. Calcium, magnesium, and other things are sequestered, and they're used for the survival and maturation of that parasite. Miltefosine, it's believed-- it's not fully understood-- acts at that acidocalcisomes to disrupt it, and through that mechanism kills the parasite.

So the sum of this slide is calcium. It's all about the calcium. So going forward-- don't be alarmed, please. I'm going to walk you through these-- the focus of our drug discovery efforts-- and by our, I mean this is a beautiful collaboration between Mayo Clinic, the Delhi University in New Delhi, India, and KEMRI in Kisumu, Kenya. A very nice global collaboration trying to identify new compounds that can target the calcium pore, the channel, which is unique to these parasites.

And we do a lot of work here with molecular modeling. So a lot of three-dimensional molecular modeling is done in our lab and also other labs. I also want to mention here, Dr. Tom Caufield, one of our collaborators here in Florida, also is very, very much involved in modeling and drug discovery. So we work with him as well.

But through a set of collaborations with the Delhi University, the Hansraj College in New Delhi, where there's a massive number of synthetic chemists, Dr. Brijesh Rathi and his group have worked with us to develop this compound, which we've given the name calcium. And again, please don't ask me about synthetic chemistry. It's not my forte. But this is a hydroxyethylaminepiperazine moiety, which has been designed and developed using modeling to fit inside and block and disrupt that central pore of the calcium channel. And that's really the long and short of what this drug is.

Now, if we look at this, the question is, if calcium is central to these parasites that have an intracellular life cycle, would this structure, this calcium channel, be conserved? So this was some work that was done by Yash Gupta and others from Delhi as well. Looking at a variety of parasites-- and what you see here on the left are these parasites, so Cryptosporidium, the Plasmodia, Toxo, some of the trypanosomes, and multiple Leish, many of which fall into that category of parasites that rely on intracellular life cycle.

And when we line up the protein sequence analysis of this pore-- and you'll see areas here that are highlighted. And then these little red dots on the top are particularly important areas. And these ones here are actually the interactive regions of this pore. You see a tremendous conservation in the structure across these parasites. And this is in comparison to the Plasmodium falciparum pore, which is actually what this code is over here. So this tells us that in all likelihood, this is a very important structure. It seems to be conserved. And if we can develop something, like the calcitonin moiety, that can block that and disrupt it, we may have a broad action of these compounds.

Now, of course, I'm sure many of you are thinking it would be great to knock out this calcium channel of the parasite and kill the parasite. But as you know, humans rely on calcium. We have calcium channels all over our body. Last thing you need is to give a drug that stops the heart from working. So you don't want to wipe out the host in doing it.

So we've done some work now. And these are some cell lines, HEK cell lines, which they're engineered to express the human T-type calcium channel. And what we've done is we've actually looked at the voltage gating through this pore at very, very, very high concentrations of our drug calcium. And we see no real effect on the actual amplitude or the actual flux. And we've also, actually, done this with calcium measurements as well.

So the idea using this calcium channel as a target becomes more appealing for two reasons now. One, there may be a broader based level of activity across multiple parasites. Not just Leishmania and Plasmodium, but possibly others. There's, when we actually look at the homology, less than 6% homology between this parasite channel and the human calcium channel. So again, this could be a very favorable therapeutic target.

Now, just to give you a little picture of some of the things that we do in the lab as we start to develop compounds for Leishmania. We have these wonderful parasites that were given to us by one of my colleagues, and really one of the world authorities on leishmaniasis, Dr. Abhay Satoskar. These are engineered fluorescent red expressing parasites here. So to keep it very simple, we use these fluorescence machines. Nice, healthy living parasites. Lots of red. And as you treat with drugs, whichever one that may be, it goes away. So you increase the concentration, the fluorescence goes up. So we're really just doing fluorescent readouts.

Here's an example of how that's done. And one of the areas that we work on in our lab, which I'm not going to get into much here, is not only looking at new drug discovery, but also repurposing old drugs, or drugs that are used for other things, like lansoprazole, posaconazole, things which may have effect against Leishmania. And again, when we test these, using these high throughput assays and doing fluorescence, as you increase the concentration, you can hopefully appreciate these red here. And then we start to come up with values. So this is just the basis of how we actually test these things.

And when we look at the structure, what I showed you previously was the structure from Plasmodium falciparum. And now these are from Leishmania donovani, the Leishmania version. And you see where miltefosine-- remember that drug that's used as a calcium antagonistic effect. Calcium, which is our putative molecule here, fits very nicely in this same binding pocket, which is this calcium voltage-gated channel. So again, we believe that there may be some targeting effect.

And by the way, when we actually tested these drugs against both Plasmodium falciparum and Leishmania donovani and others, we do get IC50s in between 75 to 100 nanomolar range, which is quite good. And we've also shown activity against multiple stages of the malaria parasite, both the hepatic stage and the erythrocytic stage.

Now, what I want to show you in the next slide-- and I always say to our students, when you do science, you got to have pretty pictures because, eventually, that's what gets you on the cover of a journal. But here's some interesting confocal imaging, and these are the Leishmania parasites. Remember, they're intracellular, so these are inside the macrophage. These are amastigote forms.

And remember, I mentioned that we have the red fluorescent ones, so there's red. Nuclei, purple. Green is actually this flow-form calcium stain. So this stains for calcium. And when you merge all of these, you get this lovely-looking cell here, which has all the components. But pay attention to the green because that's what we're going to be watching here.

So what I'm going to show you now is actually a live video image of these cells. And here's what's going to happen. When I click this, you're going to see a macrophage. Inside of it are these amastigotes. Remember, green is calcium. Lots and lots of calcium stored inside those amastigotes, those acidocalcisomes, those structures.

And this is really what's going to be happening. Before the drug treatment, there's not going to be a whole lot of green. We're going to give the drug the calcium. And we give about 10 micromolar concentration. And then you're going to see a burst. So what it's doing is it's disrupting that calcium channel and the cells are leaching their calcium. Remember, that's their survival mechanism. We are spilling it and that's how we kill it. So I'm going to run this here.

And so here you're seeing now before treatment. And right about now is the treatment. So there you can see in the center, this is actually the amastigote. There's another one up here. There's another one. As the drug is working now on that calcium core, and this is really within seconds, you see this leaching of the calcium as it comes out into the cell. And you get a picture that looks like this, and by this time, these things are dead. So the idea behind this is to target and to have that cell expel its calcium.

And this is from a recent publication, Pharmaceutics, 2022. There's a lot here. I'm just going to cut to the chase. This is the same thing that was done with Plasmodium falciparum. What I showed you previously is Leishmania donovani. So this is the Leishmania parasite. What's shown here is Plasmodium falciparum.

Again, treating. Remember, donovani, we're looking at macrophages. Falciparum, we're looking at red blood cells. These are parasitized red blood cells, which have calcium within them. And as you deliver the drug, here calcium in a 200 nanomolar concentration, you can trace the amount of intracellular calcium rising significantly here. These are all significant. And what one can see, again, this burst. And this isn't a video but just live images of the cells. So again, the role of using these things against calcium.

So I'm going to summarize, at this point, that calcium being one of these putative drugs. There are others that are in the pipeline. But the goal is to target that specific calcium pore, which is conserved across these parasites, that has enough of a distinct structure from humans that we can potentially use this as a therapeutic target, and to go after the vulnerability of the parasite, which is its ability to store.

And I'm going to finish off. I want to just show you a couple of pictures. This is Yash Gupta, who is our postdoc, who is currently now at Penn State. Dr. Prakasha Kempaiah, my colleague who has been instrumental in running the drug discovery lab here at Mayo. Some of our students. This is our colleague, Dr. Kofi in Kenya. And some of our undergraduate and PhD students over here. This is the site in Kenya, in Kisumu, sitting on Lake Victoria, right here, and the location where much of this work is done in, addition to Delhi. So as I said, all of this is made possible because of the global collaboration and network of the people that work on this.

And so to wrap all this up, I want to leave you with the thought that we're dealing with parasitic diseases that are changing for many, many reasons, all the way from climate change to human activities, parasites, the costs of controlling these, toxicity. All of these pose challenges in the backdrop of diseases of poverty. And we believe that new efforts to find new targets, such as the calcium channel modulators, hold promise, and try to show you some of the collaborations between Mayo Clinic and our international partners. And special thanks to the teams in all these locations, and the many, many students who were involved in this. So thank you for your attention, and I think Dr. Palmer is going to take over. Thank you.

WILLIAM PALMER: Yeah. Thank you, Dr. Durvasula. This was fantastic. Really interesting points made about the current treatment options and the limits of those. You mentioned briefly some comments about vaccination. Can you comment a little more on vaccination options for these types of parasitic diseases?

RAVI DURVASULA: I'll mention to you that, at the moment, just to be blunt and to summarize it, there are no vaccines that are currently being deployed. The circumsporozoite vaccine for Plasmodium falciparum, I call it the Holy Grail of infectious diseases. It's been under development for decades. The funding for it has been spectacular from many, many organizations. Still not showing widespread protective efficacy, largely because, as I mentioned with Plasmodium, that diversity and trying to knock out those specific antigens are not easy.

But the circumsporozoite vaccines, keep an eye out. There's still ongoing trials on those, but nothing yet. Leishmania is far, far behind. And the current vaccines involve using CRISPR/Cas mutations of Leishmania major to knock out what's called the centrin gene, which is essential in calcium metabolism. That is now showing some protective efficacy in animal studies, and this is work that's been going on in Africa. But still a little bit more on the horizon. So suffice it to say that we still rely on medications and barrier protection, like bed nets, et cetera, and arthropod mitigation to control these diseases.