42nd Annual Killian Award Lecture—Stephen Lippard
STEVEN HALL: Good afternoon, and welcome to the 42nd Annual James R. Killian Jr. Faculty Achievement Award Lecture. I'm Steven Hall, chair of the MIT faculty. And before we begin, I have two requests. The first is to make sure that your cellphone and any electronic devices that you have are turned off. And the second is, it is crowded and there may be some late arrivals. So if you're at the end and there's seats in the interior of the row, if you could please move in, that'd be greatly appreciated.
The James R. Killian Jr. Faculty Achievement Award was established in the spring of 1971 as a permanent tribute to Dr. James R. Killian, president of MIT from 1948 to 1959, and chairman of the corporation from 1959 to 1971. The purpose of the Killian award is to recognize extraordinary professional achievement by MIT faculty members, and to communicate their accomplishments to members of the Institute community.
The title of Killian Lecturer is the highest honor that MIT faculty may bestow on a colleague. It is our privilege today to be addressed by this year's award winner, Stephen Lippard, the Arthur Amos Noyes Professor of Chemistry. As stated in the citation from the Killian award committee, Professor Lippard is widely acknowledged to be one of the founders of the field of bioinorganic chemistry, a field concerned with the role of inorganic molecules, especially metal ions in their complexes and critical processes in biological systems.
His pioneering contributions include understanding the mechanism of action of the clinically important anti-cancer drug cisplatin in the rational design of new variants to combat the problems of cytotoxicity and resistance. Among many achievements, Professor Lippard's groups discovered the first metal complexes that interpolate into DNA.
Professor Lippard completed his Ph.D. in chemistry at MIT in 1965. And shortly thereafter, joined the faculty of Columbia University in 1966. He's been a member of the MIT faculty since 1983, during which time he has trained many young scientists. He's a teacher of freshman chemistry, and has mentored more than 100 Ph.D. students and an even greater number of post-docs.
He served for 10 years as head of the Department of Chemistry. Professor Lippard has published more than 800 peer reviewed papers, recorded nearly 30 patents, and co-authored the definitive textbook on bioinorganic chemistry. Professor Lippard has received numerous awards for his achievements, far more than I can list in this short introduction. I'll mention just a few.
He's been elected to the National Academy of Sciences, the National Institute of Medicine, and the American Academy of Arts and Sciences. He was recently awarded the 2014 Priestley Medal of the American Chemical Society, which is that society's highest award. and in 2004, he received the National Medal of Science, the nation's highest honor for scientists and engineers. So it's with great pleasure that we recognize Stephen Lippard today with the James R Killian Jr. Faculty Achievement Award.
We have a certificate of citation for you. And let me read the citation. This certificate is in recognition of groundbreaking work at the frontiers of inorganic chemistry, leading to improvements in human health and conquering of disease. And for having taught, mentored, and trained a generation of scientists at MIT. So congratulations.
STEPHEN J. LIPPARD: Thank you.
Thank you very much. It's a great honor for me to be here. It's actually very humbling to be elected by one's peers to this honor. It's an unbelievable list of MIT faculty, past and present, and men and women. And it's just great. Some of them are in the audience. And so it's a special pleasure to be here.
I was a student, a graduate student, when Dr. Killian was chair of the corporation. And I did know him. I guess I've spent more time in Killian Hall listening to music than I actually spent with him when I was a student. But it's just a wonderful honor and I'm greatly appreciative. And I thank all of you for coming out to listen. So I also want to thank my sons, Josh and Alex, who've come up to be here.
So we work at the interface of inorganic chemistry and biology. So metals and biology, the two major areas here. One is the understanding of existing metals which function in much of biology. And the other is the introduction of metals as probes and also as pharmaceuticals.
And it's in the last area that I'm going to be discussing exclusively today, talking about understanding and improving platinum anti-cancer drugs. And this work has been supported for a very long period of time by the National Cancer Institute, to whom I'm very grateful. And also in part by the Koch Institute, which we have been an associate member of for some time.
So there are basically two major themes I'm going to talk about today. One is understanding, the extent to which we understand something about platinum compounds. And I'm going to talk about the discovery that platinum compounds can cure cancer. Not something that's particularly an obvious thing.
DNA is the cellular target. And the structure of the adducts that platinum makes on DNA. As well as the functions that are affected. And from that understanding, the ultimate goal was to try to see if we could make improved versions of the compound. And I will talk about dual threat, so-called dual threat constructs to target cancer cells and to try to get around some of the problems that one sees in conventional chemotherapy.
Talk a bit about the strategy for delivery of these compounds in vivo. And then talk about a new class of so-called monofunctional functional compounds, which we think have particularly important potential for drug development. And I'll say a bit more about how we intend to go about that in the near future.
So the discovery that platinum compounds can cure cancer came about when a biophysicist by the name of Barnett Rosenberg, working at Michigan State University, carried out an experiment in which he put E. Coli cells growing in culture in electric fields. And the reason he did that was because the lines of force of electric fields reminded him of a high school biology textbook picture of cells dividing.
So we have physics and biology. I've always said that's kind of a dangerous combination. But understanding requires chemistry, so I have to give a little call out to my colleagues from the department. So here's a picture of Barney. And actually, what happened was, here is electron micrograph of the E. Coli cells growing. And here's what happens when you turn on the electric field.
And it was extremely puzzling. And so after a couple of years, they finally discovered that it had nothing to do with the electric field, this filamentous growth. But a small amount of platinum that was used to supply the electric field leached off the electrode and reacted with ammonium chloride in the buffer, forming this very simple 11 atom compound. The cis-diamminedichloroplatinum(II), later called cisplatin, has no carbon atoms in it, no honorary carbon atoms. And it turns out, that compound is a cure for certain forms of cancer.
And this is the experiment that Rosenberg did. He tried to get the National Cancer Institute and others to carry out trials, human clinical trials, but was very unsuccessful with that until he produced data of this kind. He took a mouse, implanted a sarcoma, and a tumor grew. After three weeks, the mouse died without any treatment. But a single injection of the cis-isomer of diamminedichloroplatinum produced significant necrotic tissue. After about two weeks, the tumor became smaller. It was groomed off and the mouse ultimately lived a normal lifespan.
At that point, the National Cancer Institute got very interested. And it supported a wide range of clinical trials across the country, where they worked on safety, efficacy, and looked at particular types of tumors that were responsive. And of course, cisplatin itself was a known compound. It had been around for quite some time. It was discovered by Pyrone in the mid-19th century. This 1965 discovery of cell division that led to the antitumor activity in 1969 was followed up by FDA approval only about 10 years later.
Now, it is to Rosenberg's credit that he made the connection between filamentous growth and antitumor activity. He knew that agents that caused DNA damage did bring about filamentous growth. That is growth of the cells without cell division. And that was the reason that he took it into the animal trials.
And so now, by today, cisplatin and two derivatives, one called carboplatin, where the chlorides are replaced by the cyclobutanedicarboxylate. And this compound, known as oxaliplatin, where the amines are replaced by the RR isomer-- for the organic chemists, 1,2-diaminocyclohexane oxalate, which is used for colorectal cancer.
These three FDA approved compounds are very common in clinical use. They've been in use for a very long time. And almost all of them are off of patents, so a lot of generics used. And today in 2014, actually, more than half of all cancer patients receiving chemotherapy are now treated with platinum compound, which is an extraordinary thing for an inorganic chemist working at the interface of biology, medicine, and chemistry.
So the question is how do they work? That's a very simple question, but it has a complicated answer. And I would say, like any mechanism in science or in chemistry, you never really prove a mechanism. You just come up with an idea that tells you how it may be happening and wait for somebody to say, oh no, you're wrong. There's another, better explanation. Fortunately that hasn't happened, but if it does, that would also be good fortune as well.
So the focus has been on DNA as the cellular target. And I want to talk a little bit about that and our work on the structure of platinum DNA adducts. So the human cell, from a chemical point of view, is incredibly complicated. Not only because of its different constituents, but because it's constantly changing. And so certain antibiotics, like vancomycin, for example, Merck scientists tell me that this so-called drug of last resort for bacterial, for infection, was actually-- the bacteria became resistant by inventing five totally new biochemical pathways to synthesize themselves around that drug.
And so when we try to understand and begin to look at how any outside chemical can work in this environment, it's a daunting task I found out. I didn't know that at the beginning. We had done a lot of work in the interaction of metals with nucleic acids, because we had spent some years-- and this is when I started at Columbia-- trying to sequence DNA in an electron microscope by attaching it to base-specific sites. Which we succeeded in doing by putting sulphurs at base-specific sites, quantitatively in long stretches of DNA. 1,000 to 10,000 in length.
But we couldn't actually read those beads on the string in the electron microscope because a little too much energy caused blurring of the images, although we certainly tried hard. But with that background in DNA, I thought this would be an interesting problem to study. And I want to start by giving you a little bit of an overview of what happens, and then I'll focus specifically on the DNA.
So there are really, overall, four stages, broad stages, in the cisplatin mechanism of action. One is passage through the bloodstream. It's given as an intravenous injection. And it has to encounter the tumor cell microenvironment and then get into the cancer cell, which it does by passive transport, and possibly by piggybacking on the copper transport. There's reasons for that which I won't have time to talk about today.
But the key thing is that in blood, where the chloride ion concentration's about 0.1 molar, the chlorides are kept on to some extent. Whereas in the cytoplasm, the chloride ion concentration is much smaller. There's an activation and they get lost in a stepwise fashion to produce cationic platinum compounds, which go to DNA, and bind to the strongest electron donating ligands in the DNA, which are the N7 positions of the guanine and adenine basis, forming cross-links because there are two places that it can bind. And then there's a cellular response, which I'll talk about later. And that's incredibly complicated. Thousands of papers a year talk about the cellular response to platinum compounds.
Now, why do we think DNA is the target? Well, for one thing, if you take cells of diminished ability to repair DNA, they're hypersensitive to platinum compounds, particularly cisplatin. And if you want to talk about going all the way to patients, patients who have received platinum infusions as chemotherapy have significantly higher levels of platinum DNA cross-links on the so-called buffy coat, or DNA-containing cells of blood as determined by antibodies that are specific for those particular adducts.
And patients that do not have high levels of platinum DNA adducts do not respond very well to platinum chemotherapy. So all the way from the cell on into the patient being treated with platinum, there's very strong evidence, I think, that DNA is the right target.
Now, we and others looked at some detail at how the adducts on DNA were made, and in particular, the relative amount. So about 90% of all adducts of cisplatin are intrastrand GG cross-links. So if we talk about DNA as having a Watson strand, or a Crick strand, the adduct is making a cross-link on the Watson strand. And the 1,3 cross-link with an intervening nucleotide-- so two guanines with an intervening thymine, maybe about 5% or so, and a smaller number of interstrand cross-links where one links between. So for cisplatin, for example, these are some numbers. And for the carboplatin, they're slightly different.
So the first thing we wanted to do as chemists was to learn something about the structure of these adducts. I mean, if we were the size of the atom, this would all be a visual art. But a lot of chemistry is directed at learning the three dimensional architecture of the micro world and smaller. And so the first thing that we were successful in doing-- this was the work of the graduate student Suzanne Sherman in 1985-- was to synthesize and crystallize deoxy pGpG, a dinucleotide, where platinum binds the N7's, and the two amines are here.
And you can see there's a significant bend at the position of the two guanines, which in a normal double-stranded DNA would be stacked on each other and in a series of parallel planes. And it took us about a decade, because of the difficulty of synthesizing and crystallizing, to get the structure of cisplatin on double-stranded DNA. That was done by Tricia Takahara. And there you see the platinum in the major groove of the DNA where the N7's of the guanine are. Again, making this cross-link.
The picture is very similar to that of the simple dinucleotide. But we can see that it causes a substantial bend in the DNA duplex. And it opens up-- and that's in the major groove of the DNA-- opens up the minor groove. And it opens up a hydrophobic surface to which proteins like to bind.
And even later, we were able to get the structure of the diaminocyclohexane platinum on double-stranded DNA. The diamine platinum is the adduct that's made by cisplatin and carboplatin. With this one, the diaminocyclohexane, we now have the adduct that's made by oxaliplatin. So we now know the structures of the three FDA approved compounds on DNA.
And it was very similar to what we saw for the cisplatin adduct. The one difference being that we could tell why the RR isomer was important in the activity, because there's a specific stabilizing hydrogen bond made with this amino group, with the oxygen atom of the guanine on the 3 prime side of the oligonucleotides on the Watson strand. And in the other isomer, if you have an S-configuration there, you can't make that hydrogen bond.
Now, we come to a much more difficult question, although those were difficult enough at the time. And that is, what are the cellular functions that are affected, and how do those cellular functions lead to the cancer activity, and in particular to the discrimination between normal cells and cancer cells? And a very important paper, at least in my thinking, was one published by Alan Eastman and his colleagues in 1988. It had a couple of papers, actually. Demonstrating that cisplatin arrests the cell cycle at a particular point. That is, just before it undergoes cell division or mitosis.
And so what do he proposed was that it was the inhibition of the transcription of the DNA, that is the conversion of DNA to RNA, which then ultimately makes proteins, that blocks the expression of the enzymes and proteins that are necessary to produce the mitotic spindle. And also, by the way, explains the Rosenberg result of filamentous growth. Although we have to remember that Rosenberg was working on bacteria and cisplatin killing bacteria is not a way to solve cancer. But cisplatin works on humans.
And by the way, I didn't mention-- I meant to mention this earlier. Just to give you an idea of how good it actually is. For testicular cancer, which used to be the leading cause of death of males aged 20 to 40, and would be today, it is essentially a cure. And the poster child is Lance Armstrong. He's not quite as popular a poster child as he once was.
But he had stage four metastatic testicular cancer. It was in his brain. And he had a complete cure, complete remission. Hasn't come back as far as we know. And many, many, many cases like that. In fact, the statistics say-- cancer statistics say there's about 9,000 new cases of testicular cancer each year. And a very small number of deaths-- well under 100.
So what is transcription and how do we think about platinum blocking transcription? And can we actually get evidence for it? After all, the Eastman paper didn't produce direct evidence. It was a hypothesis. And Ryan Todd, a former graduate student, and I described this in a journal known as "Metallomics" recently. And I want to focus specifically on a road block to RNA polymerase.
So here's an example of an experiment carried out by Karen Sandman in the lab, which demonstrates very graphically how transcription is affected. So here we have a construct in which we have two fluorescent dyes-- a coumarin, which fluoresces in the blue, and a fluorescein, which fluoresces in the green. And it's linked by a beta lactam, like the one in penicillin.
And this molecule is protected. And when you put it into a cell, cytoplasmic esterases in the cell will cleave off the protecting groups. And this molecule will fluoresce. So if you shine 409 nanometer light on the molecule, the coumarin will absorb. But then via a process known as FRET, or Fluorescence Resonance Energy Transfer, the photon will be transferred from the coumarin over to the fluorescein, which emits at its own characteristic wavelength of 520 nanometers. And the cells will look green.
Now, if you put into those cells an enzyme known as beta lactamase, that enzyme will cleave the linkage between the coumarin and the fluorescein, so that when you shine 400 nanometer light on the construct, in the cells they'll look blue. But if you have an agent such as platinum, which can block transcription, that is the conversion of the beta lactamase, DNA into the RNA and then ultimately into the protein, it will not be able to cleave because it won't be expressed. And the cells will stay green. And you have enzymatic amplification, which allows one to detect even very low level of gene expression.
And here's the experiment. These are the control cells not treated with platinum. They're blue. Here's cells which are treated with 40 micromolar cisplatin, which are green. And you can clearly see transcription is being affected. In fact, we used this screen one summer to look at the series of potential new platinum compounds that we had made by robotic synthesis, along the lines of what is known to be effective. Namely compounds that had cisamines and good leaving groups.
And I had an undergraduate, a UROP student, who made with that robot about 4,000 compounds over the course of the summer. And my post-doc tested them somewhat manually. And there were four that turned out to be hits. And all four of them had been tested by various drug companies as potential anti-tumor platinum compounds.
Here's another way you can look using an RNA polymerase, an enzyme isolated from T7. And so in this experiment, what we've done is we have taken the RNA polymerase and we have put it onto a magnetic bead. And we have a Watson strand, a Crick strand, and then we have a small piece of RNA as sort of a starting piece. This is one way in which you can carry out transcription.
And then we anneal that to an artificially prepared oligonucleotide-- double-stranded oligonucleotide, containing at a site-specific platinum GG or other cross-link. And so we bring these two together and we ligate them together, paste them together. And now we carry out what's called a transcription walking experiment, where we add nucleoside triphosphates.
If we have a radio label at the starting RNA strand, we can watch the RNA strand be extended, which is transcription. If the platinum blocks the transcription and we run the RNA molecule out on an electrophoresis gel, at the end of the day, we'll see 62 nucleotide piece. So relatively fast moving piece in the gel. Relative to if we had no platinum there, where we're able to bypass the adduct and go all the way to the end, it would have 133 nucleotides.
And here the data. I won't show a lot of data, but this is an example. It's very clean. You can see increasing amounts of the intrastrand cisplatin GG cross-link, or the 1,3 cross-link, or the diaminocyclohexane GG, or 1,3 GTG intrastrand cross-link leads essentially to the complete block of transcription in this particular assay.
OK that's T7 polymerized. What about in humans? What about in mammals? So if we can take mammalian cells, in the mammalian cells, we can put in an expression vector that is a piece of double-stranded DNA which we put into the cell by a process known as transfection, it will go into the nucleus of the cell. And some fair amount of it will actually be transcribed into messenger RNA. And then ultimately translated into a gene that we put into this vector, which is known as Gaussia luciferase, which has the very interesting and nice property that the cells will extrude that.
And if you let that be extruded in the presence of an appropriate substrate, it will turn on a very bright yellow fluorescence. And so you can very nicely see whether or not you're transcribing that particular gene by simply looking with a luminometer at fluorescence that comes out of the cell.
Now, if we repeat that experiment, but we take a platinum compound and we add increasing amounts of the platinum compound to the probe prior to putting it into the cell, what we can do is we can look at cells which are either able to repair or not repair the adduct. Now, I brought a new verb into the discussion, which is repair. And it's a very important verb.
So far I've been talking about transcribing as my verb, but running against that is the fact that cells are able to repair damage on their DNA. And in this particular experiment, we're looking at cells that are either able to repair XPF corrected or not able to repair. And then we actually looked to see whether it mattered whether they were synchronized or not. In other words, were they all in phase in the cell cycle or not? Turns out it doesn't matter, so I won't make any more out of it.
Transfect the probe and collect the cell media at various times. Remember, it's the media around the cells that is going to fluoresce when we put the appropriate substrate on. And assay for this GLuc activity using our luminometer as an auto injector. When I bought that, my post-doc was incredibly happy. It saved him many, many days of work to do the experiments.
And we find the following types of experiments. So if we look at a repair deficient set of cells, XPF-- I'll get to what XP means in a few moments-- and we take, let's say, a certain amount of platinum per plasmid molecule-- let's say roughly 10 platforms per plasmid-- we see that with increasing time-- and so each one of these little diamonds has a different color-- going from an eight hour treatment all the way up to 48 hour treatment, we see that the transcription levels rise as we increase in time.
So here the transcription is knocked out very much after only an eight hour period. But after 48 hours, there's a lot more transcription. And recovery with time indicates that there's repair. Certainly within the first 24 hours. 48, it gets a little more complicated because the cells have divided.
Now on the other hand, if we look at a series of cells which have been corrected, and they're able to carry out nucleotide excision repair, we find that there's a much greater amount of repair. And the recovery with time indicates significant repair compared to limited repair. And that gave us a clue.
We know that cancer cells are inherently deficient in repair. And so already then, is if there is a distinction in the amount of repair, that is a good reason for thinking why, with transcription inhibition versus deficiency in repair, the compound might be active as an anti-tumor agent.
Now, these are experiments where we had the pGLuc templates globally platinated with cisplatin. It just went everywhere. So we didn't always make intrastrand GG cross-links. We made the minor adducts. We made so-called monofunctional adducts. And the transcription levels being higher in the corrected cells indicate that those cells more readily bypass during transcription.
So we repeated it using a site-specifically platinated DNA. Now, site-specific modification of DNA is a really high call. And I have to thank my colleague, John Essigman, who I met shortly after I came to MIT from Columbia. He had done a lot of really beautiful work on site-specific modification of DNA. And we learned a lot from him, and we published a lot of papers together and had a great time looking at specific adducts on DNA. And the intrastrand cross-links are certainly ones that we wanted to look at.
So here is the pGLuc vector. For those who are biologists, we have a promoter, we have the reporter gene. We put the platinum in between the two. Site-specifically platinated. Removed the origin of replication so we didn't replicate out the platinum. And in particular, with a little bit of experimentation, we discovered the best place to put it to get good signals was between the transcription start site and this sequence that directs the translation of the messenger RNA.
So the inventory of site-specifically platinated probes included the intrastrand GG, the diaminocyclohexane 1, the 1,3, and we've also made interstrand-- much more difficult task, but we have it and published on it. And so we could look at how the different adducts were repaired.
And in this particular set of experiments, we use a different set of cells. We use colon carcinoma cancer cells, P53 wild type or mutant. These were mismatched, repaired, deficient. These were mismatched, repaired, proficient. This is the unplatinated plasmid. And these are the platinated plasmids.
So you see very strong inhibition of repair over a period of time. Sorry, inhibition of transcription over a period of time. And very little increase with repair. Mismatched repair does not remove the major cross-links of cisplatin on DNA, which is why those experiments look different from the ones using the XP cells.
Now, this method is applicable to study many cellular contexts and many different types of compounds other than platinum. And we envision a potential application in personalized medicine. Possibly, if at some point down the line, when we're able to get tumor samples from patients in a short period of time-- this is a very fast experiment-- one might be able to take a look at different types of platinum compounds, maybe in conjunction with other things that we put on the platinum, which I'm coming to shortly, to see whether certain people might be able to respond better than others to certain types of platinum-based treatments.
Now, I've mentioned repair. It's time that I tell you a little bit about nucleotide excision repair. So this work was work that we carried out in collaboration with Aziz Sancar at the University of North Carolina, who is a really wonderful colleague and somebody who taught us a great deal about looking at NER. And so all of our genomes are damaged every day.
It's estimated that there's at least a million mutations that are made every day, or damage events that could lead to mutations. And we have repair mechanisms that can take it out. Whether it's walking out in the sun. And we have some today in Cambridge, finally. Or drinking milk and getting a little potassium 40. There's a lot of mechanisms that we have that allow us to repair the damage on the DNA.
And the one that takes out the intrastrand cross-link of cisplatin, essentially exclusively, is nucleotide excision repair. And the way that works is the following. If we have damage on the Watson strand, there's a series of enzymes and proteins which recognize it, and then cut out a piece, which I show down here with the damage on it. It's about 30 nucleotides in length.
It cuts first on the 5 prime end, and then subsequently on the 3 prime end. The cut on the 5 prime end is the first irreversible event in the process. And then you have a gapped DNA duplex, where if you've taken it out of the Watson strand, then the Crick strand has the correct sequence. You can then synthesize against that correct sequence with nucleoside triphosphates and make the repair patch. And the repair takes place.
So the experiment is set up in the following way. We take cell extracts that are repair proficient and repair deficient, and we put a platinated DNA duplex into the cell extract that we synthesize ahead of time. It's about 153 nucleotides long. And if the damage is placed on the Watson strand, when we put this piece in-- let's say it might be 50 nucleotides or so-- we will be sure to have a radio label placed as a phosphate group here.
And so, for example, here is a strand of 156 nucleotides that were put together with this particular sequence. And the platinum piece that was put in was TCTAGG with a 1,2 intrastrand cross-link CCTT. And then in a separate DNA-- this is now separate strand-- we put in TCTGTG so we could look at the 1,3 cross-link.
You make the double-stranded DNA by annealing the complimentary unplatinated Crick strand to it, put it in the repair extract, and look to see whether you get a 30 nucleotide piece-- this is a control lane-- 30 nucleotide piece in your gel. Now, because it's a cell extract, it has nucleases in there other than the ones that take out the repair damage. And so your 30 nucleotide piece is going to be cut to 29, 28, 27, and so forth. So you've got a little ladder of bands.
Now, if we look at HeLa cells, which are very famous cells that are used in cell biology to do experiments of this kind, which are cervical cancer cells-- the whole story related to Henrietta Lacks-- we see that it's proficient in repair and takes out the damage for both the GG, and even better, the GTG. So we see this is a better repair substrate than the major adduct, which is already interesting.
But you see there are these lanes mark F and G. Now F and G stands for the complementation groups F and G. So these cells are derived from patients who have a terrible disease known as xeroderma pigmentosum. It's about 1 in 100,000 individuals. And they lack one or more, usually one, genetically inherited loss of a particular one of those enzymes that's involved in the repair complex.
But when you put an XPF patient's cells together with an XPG patient's cells, they complement each other. And so as a consequence, you get very good repair signals when F and G are put together, but not when either is there alone. And so we can look at the kinetics. That is the rate at which the excision occurs as a function of time. It's shown here. And the GTG is better than the AG or GG instrastrand cross-link.
Now, the question is, how does all of this translate into the killing of cancer cells? And the answer is a battle that goes on in the cancer cell, namely that of transcription inhibition versus the inhibition of nucleotide excision repair. And I have a little cartoon that will perhaps bring this home to you.
Imagine that you have a piece of DNA that's got cisplatin damage. RNA polymerase II, which is the major transcribing molecule in cells, comes along, hit's the damaged site. The stalled RNA polymerase triggers multiple cellular processes, calling out for repair or programmed cell death. So it's going to go down one of those two pathways.
And our lab has studied this. We did some work with Phil Sharp in the past, leading to some insights into this process. And failure to recognize and repair the damage in answer to this 911 distress call is what you want in the cancer cell. Because what will happen otherwise is that the stalled RNA polymerase II will be phosphorylated at the 3 prime end, and then ubiquitylated with the proteins known as ubiquitin, taken to the proteasome, which is basically the garbage can in the cell for taking proteins apart.
The cellular repair machinery will be recruited, particularly nucleotide excision repair. And recognition and repair of the damage in answer to that distress is what you want in healthy cells. And we believe that the anti-cancer activity of the platinum drugs exploits that property.
Now, one of the very interesting questions which I had originally planned to talk about today, but I just don't have time to get it all in-- and we haven't finished the story-- is why it is that testicular cancer cells are so affected? Why is there essentially a cure of testicular cancer and not of so many other cancers?
And I don't know that this is the answer, but we're tracking down a rather interesting lead right now related to a particular protein that is found in all cells. It's in an amazing protein, known as high mobility group protein, that likes to bind to the cisplatin intrastrand cross-link. And when it does that, it blocks nucleotide excision repair. And so it can sensitize the cells to the platinum, because it removes the ability of the cell to repair.
We tried to prove that over the years. And we had a data which did prove it, and we had a data which disproved it. And we published both sets of experiments. And we just didn't-- it was just very frustrating to try to get any degree of consistency.
But recently, a form of the high mobility group protein appeared in testes that was different from that in all other cells. What I think, perhaps, the variable that we were not controlling in all of those earlier experiments was the intracellular redox potential of the cell, which is pegged at a certain value depending upon, if you're a chemist or a biochemist, the glutathione concentration in the cell.
And if the high mobility group protein has a pair of cysteines, and if they make a disulfide bond, they aggregate the interaction of that high mobility group protein with the platinum. Knock it off by about a factor of 1,000 by flipping a phenylalanine side chain that's inserted into the hydrophobic notch opposite the platinum. I have to be careful here, because I might be adding back in the time that I said I was cutting out.
Anyway, the important thing is that in the testes cell, one of those cysteines is a tyrosine. So it can never make the disulfide link. And now we're working very hard to be able to test that. And the big problem that we have is there just aren't enough-- fortunately, it's a good thing, I think-- testicular cancer patients in the world to give us enough cells to be able to test this. But we're meeting soon with some colleagues at Mass General. And we hope to be able to come see whether that hypothesis is the correct one or not.
Now, in the remaining time, I want to talk about attempts to try to improve things. Now, why do we want to improve things? Well, I'm sure you're aware of the fact that chemotherapy has its toxic side effects. And in the case of platinum compounds, there's either acquired or inherent resistance. And in some cases, the tumors, despite every attempt of the physicians, are just simply too aggressive and patient dies of the cancer.
Now, that has led us and others throughout the world to look at so-called non-classical platinum compounds. And I'm going to talk about platinum IV compounds, which are so-called dual threat compounds, and so-called monofunctional platinum compounds. Now, the dual threat strategy is the following. That is, we want to take a platinum compound like cisplatin and add two additional ligands to it by oxidizing it from platinum II the platinum IV.
If we could get that into the cancer cell, it would release the extra ligand, producing cisplatin. And the extra ligand, which if it had its own anti-cancer activity, could serve as a dual threat to try to knock out the cancer cell before it could become resistant or otherwise escape the chemical assault. The other possibility is to package these in nanoparticles that controlled the release of the platinum that platinates the nuclear DNA and operates by the mechanism I described, and/or produces another unit, x in this case, or l in this case, conveys an alternative biological activity.
And our approach was to build a platinum IV compound, which you can do quite easily chemically by oxidizing to the dihydroxide and then attaching a group R. Platinum IV compounds are more inert than platinum II, which allows good synthetic manipulations. And in one case, actually led to a compound that could be administered even orally. Didn't pass the FDA, but it came very close.
And different pharmacological and pharmacokinetic properties-- the spectrum of activity, the R group provides a dual threat. Reduced side effects, the R group could target a cancer cell versus a normal cell. Drug resistance, which can alter the mechanism of uptake, which cells sometimes block the uptake into the cell, which I haven't talked about. Or the compounds could reduce rapidly to potent platinum II compounds.
Now, the first dual threat compound that I thought about came as a result of reading Nature magazine on an airplane flying back to Boston from a trip that I was taking. And I was reading about a molecule known as dichloroacetate, or DCA, the potassium and sodium salts of which had been used to treat lactic acidosis. But cancer patients found that as an experimental cancer drug, it could shrink tumors in animals.
They wanted to do clinical trials in humans, but there was no incentive to do that. There was of a lot of discussion about this in the end of 2000, first decade. Because it had been around for years, its structure couldn't be patented. The drug companies weren't interested. And a lot of the work was done in Canada. And this molecule is still undergoing clinical trials. It's now in phase three in Canada. I looked recently, there's nothing new.
So the plane hit the ground in Logan. I was on my cell phone. And I called my post-doc, Shanta Dahr. And I said, Shanta, it should be very easy for us to attach the dichloroacetate to a platinum IV derivative. See if you can't make this compound.
And literally, before I got to the lab the next day, she had made it. That's how easy it is to make that compound. And we called it mitaplatin. I wanted to call it mitoplatin for mitochondria, but that had been used by I won't say who. Just a very odd thing. So I made up it mitaplatin.
And then one of post-docs says, I really should call it MIT-aplatin, just in case it--
And I'm willing to fly the flag. I don't have a brass rat, but I'm willing to fly the flag. All right. So mitaplatin, you see, would go into a cell. It would be reduced to platinum II, producing dichloroacetate. Now, what does dichloroacetate do? It takes advantage of something known as the Warburg effect. That is it's known for some time that cancer cells, when they first get going, have somewhat oxygen deprived environments.
And so their mitochondria don't work by the usual oxidative phosphorylation affects converting glucose into ATP. And so they go over and they use another mechanism, similar to what yeast do to anaerobically use-- to convert the glucose. And it turns out that dichloroacetate inhibits a key enzyme in that process known as pyruvate dehydrogenase kinase.
And so when that happens, the potential across the mitochondria, the cell potential, substantially decreases, leading to the release of cytochrome c, which is involved in apoptosis, or the programmed cell death pathway. As well as something known as AIF, the Apoptosis Inducing Factor, which goes over to the nucleus and helps the platinum DNA that's stalled here produce the killing effects themselves.
And so we decided to look at the effects of mitoplatin on cancer cell mitochondria, and we discovered it indeed changed the membrane potential. It released the AIF in cytochrome c. And most importantly, the platinum compound got in there, made cisplatin, or an analog, got onto the DNA, and formed the intrastrand GG cross-link as confirmed by using a monoclonal antibody known as R-C18, which I'll show a little bit later as well. Which is very specific for the intrastrand cross-link made by cisplatin.
So this is kind of a cartoon summary of what it does. And then if we look at the effect against cells, and NTERA-2 testicular cells, HeLa bone, platinum sensitive or non-sensitive cells. We found it was a very potent platinum IV compound.
This is really a proof of principal type idea. We don't think that mitaplatin's actually going to find its way into patients, or necessarily should. But it shows the dual threat, the way in which you can attack two different types of processes in the cell. Which I think would make it much more difficult for the cell to become resistant. And hopefully, lead to clinical success.
Now, the other way, of course, is to deliver platinum drugs in vivo by strategies that involve nanocarriers. And we've been working on this for some time. Many of my colleagues at MIT, including, I'm sure, some in the audience working on this. The idea being, using nanotechnology, to take platinum compounds, or other drugs for that matter, selectively into cancer cells. And perhaps protect them from reactions in the bloodstream that might inactivate them.
One of the ones we looked at very early were single wall carbon nanotubes, which we did in collaboration with Hongjie Dai's group at Stanford University. I call them long boats because he sent us these long tubes that reminded me of the Viking long boats. And I said, Hongjie, how many oars do you have on your boat?
And he said, well, we have about 80. And we had just, at that time, quantitated the amount of platinum that we could put on those oars. And it turned out to be 78. And I said, OK. That stuff is good. I can work with this material. I was very happy about that.
So here's an example of where we actually, using platinum atomic absorption spectroscopy, proved that we have 80. And we put folic acid on one of the two axial positions of platinum. So we oxidized the platinum for dihydroxide. We put a succinic Make acid on both ends, so we make the disuccinate. And one of them we turn into a carbamate with linker chains, ethylene glycol linker chains, which gives water solubility. And out to the end, we put folic acid.
And folic acid is known to target folate receptors on cancer cells. And so when we treat those cells, they will bind to the folate receptor, be internalized, and maybe direct the platinum compound, which would be released inside the cell as cisplatin specifically to that type of cell. This is the immunofluorescence study that indicates, again, using this monoclonal antibody R-C18, that in that experiment, we make the 1,2 intrastrand cross-link.
And then if we look at the cytotoxicity against the different sets of cells, folate cell receptor positive versus-- that's these types of cells-- for the negative one, the testicular cell, we find that the platinum single wall nanotube construct was more than eight times effective in the folate receptor positive cells than in the negative cells. And from the clinical point of view, if you could direct your compound against cancer cells versus normal cells, that would be a very significant achievement.
Now, then at that point, I went over and talked to Bob Langor. Who wouldn't, if you're interested in doing nanoparticles? And I said, Bob, you know, this is what we've got. I would like to take more advantage of nanoparticles. And I'm particularly enamored by your wonderful work using the PLGA-PEG, the Poly Lactic-co-Gycolic Acid.
He said, you know Steve, I've got this wonderful former post-doc named Omid Farokhzad who works with me. He's over at Harvard Medical School at the Brigham. He might be in the audience somewhere. And together with Omid and Bob, our labs have made a number of constructs where using nanoprecipitation, we've put in what would ordinarily be very hydrophilic platinum drugs with hydrophobic side chains-- this again, the work of Shanta Dhar-- into these particles.
And in fact, the three of us have co-founded a company known as Blend Therapeutics, which I'll talk about in a little while, which takes advantage of some of this nanoparticle formulation. But in addition, is very interested in these monofunctional compounds that I'll get to the few moments.
So in this case, what we did was we wanted to put something on the outside of the cell that would direct-- the outside of the polymer, rather, that were direct it specifically towards cancer cells. And so we put a so-called aptamer that was a PSMA aptamer that went after the prostate-specific membrane antigen. Which is overexpressed on the surface of most cancer cells.
And we saw that the platinum could be released over a period of time. That we could load up about 15% of the polymer by weight. And we had a fairly narrow distribution of the polymer.
And then we would take this PSMA overexpressing LNCaP cell and treat that cell line with a platinated nanoparticle, with the aptamer on it that would direct it towards the receptor on the cell. And in this particular case, we also co-encapsulated in the middle of that polymer a cholesterol that had a fluorescent label so we could watch what happened as it went in the cell.
And the fluorescent label showed that the cells were taken up by endocytosis in the PSMA-- Prostate-Specific Membrane Antigen-- over-expressing LNCaP cells. Using, again, this same R-C18 antibody, we could prove that the platinum was getting out of the particle, was being aquated, was going to the DNA, making the intrastrand cross-link. So the platinum was behaving the way it should behave delivered by the particle.
And then when we looked at the cytotoxicity profile, we found that the targeted particles were fourfold more effective against the PSMA cells, and 80-fold more effective than cisplatin. That looked good, so we decided, OK. Let's take it to the animal. So I have to tell you, that's a huge difference in drug development in going from conception to the test tube, the test tube to the rodent, the rodent to a primate, and then of course, the primate to humans, which is what we ultimately want to do in human clinical trials.
So we did rodent studies. With rats, we found the maximum tolerated dose significantly greater than that of cisplatin. That's a good thing. The blood retention in rats in the polymer was 77% versus 1.5% of cisplatin in the absence of a nanoparticle.
We did a nude mouse xenograph study, where we take a mouse that is immunocompromised. We put the LNCaP cells onto the animal. We did that dose schedule. And we found the nanoparticle was more effective at curing cancer in those mice as measured by the reduction in the volume of the tumor at 1/3 the dose of cisplatin. No recurrence after 45 days. Hey guys, let's found Blend. So we got very excited about that and we ultimately were able to do that.
Now, also related to the founding of Blend was the fallowing and last part of the summary that deals with the improving aspects. And that's the following. I remind you about the four stages in cisplatin mechanism of action. The cell entry, activation, binding to DNA, and then the downfield effects.
And what this particular line of work was, a study that we did in collaboration with a woman named Kathy Giacomini at the University of San Francisco Medical School. And Kathy was very interested in understanding how oxaliplatin got into cells. And so she asked me if I would collaborate with her, and we did.
And we looked at the ability of oxaliplatin to kill cancer cells. And compared that to-- we compared two sets of cancer cells, ones in which the organic cation transporter, or OCT, was greatly over-expressed on that cell-- because she thought that that was the pathway by which the oxaliplatin was getting in the cell-- to those cells where the organic cation transporter was not over-expressed.
And the size of these bars indicate the ratio of the ability of oxaliplatin, shown here, which has organic material on it, to kill cells if they had organic cation transporter 1 versus no organic cation transporter. That's the brown bar. Or the orange bar is if it has organic cation transporter 2 versus one that didn't have it.
Here is cisplatin, which has no organic material at all. Doesn't really care about that transporter. But oxaliplatin did. And during the course of that work, I wondered, well, how is it that oxaliplatin, which isn't even a cation, has to lose its oxalate, would be able to respond to these different levels of the transporter. So we began to look at cationic compounds that we had in our lab to see whether they would do an even better job.
And what we found was this molecule, which had been studied several decades ago by these individuals. Steve Hollis was a graduate student of mine at Columbia. And they work for Englehard Industries. And both Englehard and Johnson Matthey, these mineral and mining companies, had set up small laboratories to try to develop new precious metal-based platinum drugs. Didn't go very far, but they were interested.
And during the course of this work, Hollis and his colleagues discovered that certain monofunctional compounds were active in the Rosenberg-type experiment, where you inject the tumor in the belly of the animal, inject the compound, and look for efficacy. Now, what is this compound? This compound has one of the chlorides replaced by pyridine.
I already told you that the monofunctional compounds were not very active. And I can show you now what were the so-called early rules. The following properties were thought to be essential for observed anti-cancer activity in platinum compounds. They should be neutral. Have no charge.
Well, of course, this compound that I showed you has a charge. Cationic. It should be neutral. They should contain a pair of cis leaving groups. That compound did not. It only had chloride that left. The pyridine didn't the other two ligands were important.
And it had been known back in the early days that this compound and this compound were inactive. So it was believed that all monofunctional compounds would be inactive. But the pyridine compound had a little activity. And we thought, well, why is that?
So we began to look at it a little bit more detail. We saw it was active in animal models. It formed monoadducts on DNA. What features made it that way? So we decided that we would immediately take a look at the structure of the molecule that we called pyriplatin on double-stranded DNA.
Now, although it took us 10 years to get the structure of cisplatin on DNA, it took Ryan Todd 10 weeks to crystallize and get the structure of pyriplatin on DNA. It's not the Ryan isn't good, he's great. It's just that the methodologies improved that much over the period of time. Here's the crystal structure of pyriplatin on DNA.
It's standard, garden variety, Watson Crick, double-stranded DNA, no band. The platinum is bound to guanine, where we put it in this particular sequence. And that's its favorite binding site. The pyridine is hanging out here in the major groove. And there is no architectural change in the DNA. It's very interesting.
We then looked at transcription inhibition and nucleotide excision repair. And whereas cisplatin inhibits transcription enormously-- the blue refers to this axis-- it does get repaired to about 60% after 60 minutes by nucleotide excision repair. The diethylenetriamine compound-- see if I can get back to it quickly maybe-- this one, that's the monofunctional that led to the original structure function rules. Very poor transcription inhibitor. So no wonder it wasn't active. It wasn't affected at what we know to be, at least for cisplatin, the major activity in the cell.
So there's a lot of transcription bypass shown here. And the repair is almost nonexistent. Why is it? The cell doesn't care that it's on there, pretty much. But the pyriplatin is intriguingly in between. That is, it blocks transcription and it's repaired, but not nearly as well as cisplatin. And that's, we think, because it's not making as big a structural change in DNA.
There are sensing proteins that sense damage on DNA. And three dimensional structure is one of things that they sense. So it inhibits transcription by pol II nearly as efficiently as cisplatin and eludes nucleotide excision repair.
Then we looked at a panel of different cells. We actually looked at the NCI-60, if you know what that is. But we had our own little panel of cells-- colorectal, breast, and so forth. And you get plots like this. And what these plots are, down the middle is the mean inhibitory dose that kills 50% of the cells. And bars on this side represent cells that are better treated by the platinums. That is, they are killed at lower doses. And cells on this side means that the cells are treated worse than the mean.
So here's the pattern for cisplatin. Here's the pattern for oxaliplatin. This tells you the oxaliplatin is going to hit a very different spectrum of tumors than cisplatin. And it was on the basis of information of that kind that oxaliplatin was developed as a billion dollar drug in the first place. Being able to distinguish and differentiate different types of cancers.
Well, pyriplatin was different from either of those. So that was an interesting thing. The problem is that its potency was down by two orders of magnitude. That is, its anti-proliferative property is different from that of cisplatin because the pattern's different, but it's less potent than an order of magnitude.
So how can we improve pyriplatin? So we thought if we're right about transcription inhibition being the key factor, let's take a look at what pyriplatin looks like in a transcription complex. So we made one and we studied it by crystallography in collaboration with my former graduate student, Dong Wang, who's on the faculty at UC San Diego, and who had trained with Roger Kornberg for his post-doc.
And so we built an oligonucleotide containing a blue template strand containing the pyriplatin. We put in an RNA, the growing RNA chain. We did biochemistry as well. But in the crystal structure, here's the non-template piece of it here. And we could see that this monofunctional adduct could get into the active site of the RNA polymerase, could make the GC-based pair, but it could not translocate out of there. Which is why it was effective.
But what we also saw was that if we could increase the size of the pyridine, we might improve the potency. So we did. And my post-doc, Ga Young Park, examined a small library of different heterocyclic nitrogen ligands. And when we came to phenanthridine, we hit the jackpot. We had this compound which we called phenanthriplatin.
Because when we examined it against a series of cells-- lung, ovarian-- this is the panel I showed you before-- cervix, breastbone, and so forth. Compared cisplatin, oxaliplatin, pyriplatin, and phenanthriplatin, we can see that against this panel of lines with the IC-50 values, phenanthriplatin is substantially more potent than-- certainly than pyriplatin. Hundreds of times more potent. And more-- from 70 to 40 times more potent than cisplatin or oxaliplatin. And it retains the differential toxicity against the different types of cells.
So we're pretty excited about it. We think that phenantriplatin and compounds like it could be developed and go forward. And again, I call out Blend. Of course, there's a conflict of interest here. But the financial side of it is not what's important to me. I would like to cure at least person of cancer before I leave the planet. Many reasons for wanting to do that.
I want to finish by calling back to inorganic chemistry, which is my roots. I did my thesis here at MIT on the chemistry of the bromo renates. There were no biomolecules in it whatsoever. And I remind you that platinum is a third row transition element.
I actually, when I was department head, tried very hard to have periodic tables placed on this room. And I even got the provost at the time to agree to allow me to run a contest among the student-- but then the provost changed and we couldn't do it.
Now, here pyriplatin is a monofunctional adduct. And what is very interesting to that for an organic chemist is cisplatin makes a cross-link. When the DNA bends, if you look stereochemically, there's a stereochemical block, a structural block, to the addition of two extra ligands on the platinum. So it's very hard to put an octahedral six coordinate compound making a cross-link on DNA.
But if you have a monofunctional adduct, then all of a sudden, the space is there. And so it allows one to think about moving to the left, or west, in the third row of transition elements, away from platinum, which makes only four. Iridium likes to make six, but it also can make four, depending upon the oxidation state. And so we've been examining iridium, osmium, rhenium, tungsten, and tantalum as new potential anti-tumor drug candidates which could conceivably work by a very similar mechanism as a pyriplatin.
And I want to show you just result of one of them today. We've got some really cool results. And one of the things we want to do is to specifically go after the cells in the tumor microenvironment, like for example, cancer stem cells. Although that's controversial. But we have now some good-- very good compounds in that list that seem to go specifically after cancer stem cells. And we're very excited about it.
But I just want to talk about osmium today. And so we took this osmium compound, and osmium with two nitride and a two-nitrogen donor ligand known as orthophenanthroline, and three chlorides. So it could make monofunctional adducts, or it could make bifunctional adducts. But it could certainly make monofunctional and be accommodated in the DNA. It targets genomic DNA. And it leads to the apoptosis of cancer cells.
Very interestingly, in examining a number of compounds of this sort, my post-doc, Rama Suntharalingam, who basically invented this project, put a couple of phenyl rings on here. And it also led to apoptosis, but intriguingly, by a completely different mechanism in the cell. And that's really interesting to me, because it opens a whole new strategy, strategical way of attacking cancer cells by decorating the periphery of ligands on metal complexes and directing them to different parts of the cell, by which they could bring about different pathways.
And how we do this? I won't go through any detail, but just to say with certain biomarkers, we can prove DNA damage. With certain other biomarkers, we can show-- or with the same biomarkers rather, we can show little sign of DNA damage. And then in collaboration with Mike Hemann's lab and the Department of Biology-- there's, a nice article about him on the web today. I found that he plays the bass guitar, for the first time. I look forward to hearing that someday.
We've been looking at the-- Mike has developed an RNA signature. And looking at the osmium, we see that this compound indeed damages DNA. It leads to cell death. Whereas this bathophenanthroline with the phenyl rings is a DNA damage independent mechanism which leads to cell death.
And if you look in detail at the pathways, and we have not all of the information we want, but we have some information suggesting that ultimately, it's P53 that drives the apoptotic response. And it can be induced either by the typical pathway that cisplatin uses, or by going to the endoplasmic reticulum, through the unfolded protein response, in the ER, it could also lead to this pathway. Which could be an apoptotic response. We'll see. My colleague, Matt Shoulders is very interested in working on that. And Matt's around somewhere in the audience. And we hope to do a little more work to get some details of that pathway.
So this is a summary of what I've talked about this afternoon . And give you some idea of our understanding and hopes to improve the platinum drugs. Before I close though, I have one little retrospective that I want to share with you. And it's the following.
I want to see if I can close the loop between phenanthriplatin at the end of the story, monofunctional adducts, and the initial discovery of cisplatin. I've wanted to do this. I've convinced my graduate student, Tim Johnstone, that this would be a great experiment to try. Tim is a wonderful graduate student. He published nine papers last year. When it was time for his year end report, he said, my year end report are the nine papers. I said, that's fine. I don't want anything else from you.
So remind you, will the active monofunctional platinum compounds produce the filamentous growth in E. coli that cisplatin did? These are the Rosenberg pictures. OK? So we tested four compounds. And this was just recently published. Cisplatin, which we know produces the filamentous growth. And then we took the triamminechloroplatinum, which was the reason the monofunctionals weren't supposed to be important at all in the early days, pyriplatin, which was the first hint that they might be, and phenanthriplatin, which is really terrific, and we did bacterial expression.
Now, these aren't going to look like the Rosenberg pictures, because this is an electron micrograph. This is just light microscopy. So here's a control. Those are E. coli cells growing in culture. You treat with cisplatin, you get filaments. So we repeated the Rosenberg experiment.
You treat with a triammine platinum compound, you get no filaments. Inactive compound in humans, in mammals. Pyriplatin, you get just the first hints of a filamentous growth. And phenanthriplatin, you get filaments. Now, do E. coli tell you what's going on mammals? I don't think so. But this is just, to me, a very interesting comparison.
So I want to close by acknowledging the people whose work I've talked about today. I've been very, very fortunate in the graduate students and post-docs I've had over a long career. They've just been fantastic. And their work is the reason that I'm standing here today.
Not only are they good scientists, but they also are very clever experimentalists. And they think about things and make great contributions. And that also goes for the UROP students who have worked in my lab. I've had many UROP students, many who are first authors on papers, that have come through. Often I take them as freshman. And my graduate students have run little mini groups in my group by mentoring several UROP students, which has helped them greatly in their own independent careers.
And then, of course, without my collaborators, I'd be nowhere. I mean, without the people who have talked to me and helped us and learned. I've had six sabbaticals, and several of those sabbaticals have been basically to learn new methodologies of different kinds. Not only in this area, but in other areas we work on. So I want to thank them for all the wonderful insights that they've helped us in with methodologies. I'm a very problem directed person, which means I have to reach out to others to learn methods.
And this is my current cisplatin subgroup. You can see it's a very international group from multiple continents. As we all have to have these days. And we've got Mark down in Congress, down in Washington, is going to make sure we keep getting good funding for our international students.
So again, I just want to close by thanking all of you for being here and expressing my great thanks to the committee who voted for me. And my acknowledgement to the men and women, prior Killian lectures who've come before me. Thank you very much.
STEVEN HALL: Thank you for a terrific lecture. Professor Lippard will now answer questions. There are microphones in both of the aisles. We ask that you use the microphone so that we can hear you on the videotape. While you're making your way to the microphones for questions, I'd like to invite everyone to a reception to follow the question and answer period. It will be downstairs in the Bush Room, that direction, Room 10-105. Are there questions? OK, this is scary. Phil's going ask the first question.
AUDIENCE: Steve, beautiful lecture. Thank you very much. Thank you for the great science. Cisplatinum was one of the first, or as you showed, widely used drugs that are called a cytotoxin. But they clearly respond differently in different cells, as the testes, testicular cancer shows. So have we been able to match genotype or self phenotype in any specific way to the generality of cisplatinum response?
STEPHEN LIPPARD: Not yet. First of all, I would say all treatments of cancer are cytotoxic. And it's just unfortunate that the word cytotoxic has had a negative pall cast on it by the community. I think that we certainly have tried. So the question is, would there be resistance phenotypes, for example? Would there be certain types of ways that cells respond that we could say are tumor specific? I think that's what you're asking, really.
And we don't have enough information yet to be able to pull that out. But it's a very good thing to think about. When I first came to MIT, Bob Weinberg and I grew up together. We went to elementary school together. And we talked a lot about trying to figure out whether there might be a way to hit cells with platinum, and hit them and hit them and hit them, and look for the survivors. And say, OK, this is what's surviving. Then genetically, what are they doing?
And we did a little work. We looked at mechanisms for putting things out, but we were not able to see anything that was really a smoking gun. So I think that the best hope is really to sort of look at the human clinical samples, if you can get them, try to get them, and find out those cell types, the phenotypes of those cells-- and so I think the cancer stem cells offer a good opportunity-- that are surviving. And then try to work from that, as I'm not the only one to think of this, of course.
But I think platinum and heavy third row transition metals offer some opportunity there. But it's always a battle of killing the cell versus producing some toxic side effect to the host. And to the best we understand it now, we know what the parameters are. We just have to see if we can get them to be optimized.
And I can tell you that the people in the company are much better at doing this than we are, for all kinds of reasons. And the reason for developing the company is because I've found over the years that it was really impossible-- and it's the only company I've ever been involved in founding. It was impossible to bring somebody in from one of the big biotechs, let's say Bristol Myers Squibb, which sold all the original platinum compounds, to get them interested in taking what we had from cell culture studies and investing into what ultimately would be human clinical trials. But the startups offer a very interesting opportunity to get there. And I think we're going to get there in Blend fairly soon with our first clinical trial.
AUDIENCE: Thank you for the wonderful talk, Professor Lippard. And I agree. I wish we did have a periodic table up here, because earlier I had to go onto my cellphone to refresh myself with the periodic table.
STEPHEN LIPPARD: There's still a chance. I mean, it's empty space.
AUDIENCE: So what I was wondering, and I was kind of surprised about the work going on with the third row elements, was in terms of palladium and nickel having the same valence electron orbitals, if anyone has looked at anti-tumor compounds in those elements.
STEPHEN LIPPARD: That's a great question. And I often tell the audience that whenever a speaker says that, it means they know the answer to that question. Of course, people tried that. And they're kinetically more labile. Meaning that they're like most transition metal compounds, controlled by thermodynamics and not by kinetics. Meaning again, teaching some chemistry, that if there's a thermodynamic root to a product, it will get there.
And palladium and nickel have much more kinetic lability. That is, they'll get to that root. And platinum is more inert in the plus two state. And it's even much more inert in the plus four state.
You know, the difference between organic and inorganic chemistry is just exactly that. We would all be CO2, water, and ammonia if it weren't for kinetic stability of organic molecules. So people have tried those. So the third row elements are more inert. And some of the second row ones. And it offers a lot of opportunities.
And there have been, quite independent of anything we've done, scientists in Europe have been examining ruthenium compounds. And there are ruthenium compounds in third generation clinical trials for cancer in Europe, in patients. So they didn't come at it with the same mindset that we did related to mechanism. And I don't know whether that's going to turn out that they will behave similarly in terms of their biological profiles, but it's possible.
Now, they're a little more labile, but the particular ones they use, which are organometallic compounds, are not so labile. And they seem to be effective.
AUDIENCE: Thank you.