Marc Greenberg (Postdoc '88-'90), Chemical Biologist and Leader in Nucleic Acid Research

When did bio-organic chemistry evolve into chemical biology? As Marc Greenberg describes it, the transition was both a conceptual and technological breakthrough: before the revolutionary advances led especially by the Dervan Lab at Caltech and the Schreiber Lab at Harvard, chemists were limited by working with molecules that nature provided. But in the approach of chemical biology, chemists can make new molecules that are not found in nature, and use these molecules to probe biological phenomena at the cellular level. The result has transformed both fundamental science and translational research. Greenberg's lab at Johns Hopkins, focused on nucleic acid and in understanding DNA damage and repair, is at the forefront of these discoveries.

In the discussion below, Greenberg recounts his dual undergraduate degree at New York University and Cooper Union, and his PhD research with Jerome Berson at Yale, where he learned NMR spectroscopic techniques and focused on diradical molecules. Berson, who was Peter Dervan's PhD advisor, encouraged Greenberg to consider Caltech for his postdoc, and Greenberg describes his time in the Dervan Lab as a turning point in the nascent field of chemical biology, led by Dervan's energy and exacting standards of scientific excellence.

Following his initial faculty position at Colorado State, Greenberg moved to his current position at Johns Hopkins, where a thriving chemical biology program, coupled with strategic partnerships with the medical school, have allowed the Greenberg lab to become increasingly involved in cancer research, given its "natural connection" to studies in DNA damage. Although he describes his work as basic science, the translational possibilities are exciting, and in measured tones Greenberg conveys some breakthrough possibilities that could contribute one day to saving lives.

DAVID ZIERLER: This is David Zierler, Director of the Caltech Heritage Project. It is Friday, November 10, 2023. It's my great pleasure to be here with Professor Marc Greenberg. Marc, it's wonderful to be with you. Thanks so much for joining me.

MARC GREENBERG: Well, thank you, David. I'm happy to participate.

ZIERLER: To start, please tell me your title and institutional affiliation.

GREENBERG: I'm the Vernon Krieble Professor of Chemistry at Johns Hopkins University.

ZIERLER: Who is or was Vernon Krieble, and is there any connection to your research?

GREENBERG: There's no connection to my research. Vernon Krieble was a chemist. I understand he was a professor at Trinity College in Hartford, Connecticut. He was, I believe, an organic polymer chemist and he started the company Loctite. If you've ever gone to Home Depot, it's like super glue.

ZIERLER: For screws.

GREENBERG: Loctite was his company. He passed away many, many years ago, in the 1960s. But there's a foundation in the family name, and I know that there's at least one other Vernon Krieble Professor of Chemistry at Brown University. I don't know the connection between Hopkins and Vernon Krieble. I do believe there was a connection with Brown. And until a few years ago, I would communicate yearly in the form of a letter with their son, Robert. He was part of the Foundation, and I would apprise him of our research. But he passed away I think around the time of COVID.

ZIERLER: Tell me about some of the major research questions you've pursued in your career.

GREENBERG: Our research has been mostly concerned with basic questions concerning nucleic-acid chemistry and how nucleic acids are damaged and repaired. In some more applied projects we try to capitalize on discoveries that we've made while addressing these fundamental questions. I know we'll come back to this later, but there's a clear connection to my experience in Peter's lab at Caltech, given the overarching goals of our research.

Focus on DNA Damage

ZIERLER: What aspects of your work have translational value? And are you yourself directly connected in the clinical world?

GREENBERG: I'm not directly connected in the clinical world. We have tried and are trying to make such connections. That was actually part of the motivation for me to move from where I was to Hopkins around 21 years ago, to be at a place with a great medical school. Some of our projects, including our development of inhibitors of polymerase enzymes, which are involved in DNA repair, and also our attempts at making radio-sensitizing agents have potential translational value, but we have not yet succeeded commercially.

ZIERLER: What does radio-sensitizing agents mean?

GREENBERG: It's the idea of trying to enhance the ability of radiation to kill cells. The accepted cytotoxic target of radiation is DNA, and so DNA damage leads to cell death. However, many tumors are somewhat less sensitive to radiation, and that's because they are generally hypoxic, which means they're deficient in oxygen. It comes down to a really basic chemical issue, which is that the radiation damages DNA via radical processes, and radicals react with oxygen. In hypoxic tumors, which are often solid tumors, where there's a deficiency in oxygen. The idea is to enhance the effectiveness of the radiation to get around this. Hence, the molecules that one might produce for this are radio-sensitizing agents.

ZIERLER: At a dinner party, when people might ask you what kind of chemistry you do, what do you tell them?

GREENBERG: Nowadays, I try to keep it within a sentence or two, and I say, "I try to understand and exploit how our DNA is damaged and repaired."

ZIERLER: I wonder if you could take me on a verbal tour of your lab. What does it look like? What are some of the most important instruments?

GREENBERG: Our lab is divided into two components. One would be a conventional organic chemistry lab, where you would make and study small molecules, and the other lab is mostly biochemical. That's where we have a lot of the instrumentation that we would use to analyze molecules. It would look like a typical biochemistry lab with pipettes, and we still use radiation, so there would be radiation shields to protect you just like in Peter's lab when I was there more than 30 years ago. We have several kinds of chromatographs, such as HPLC, and UPLC, and FPLC, which are used for separating and detecting different kinds of molecules. We still have our own oligonucleotide synthesizer, which is somewhat rare. They're not made by many companies these days. We need it because we make unnatural, non-native nucleic acids. We also have a mammalian cell culture facility because we've gotten more involved in the past several years doing our own cell biology experiments.

ZIERLER: Have you gotten involved in startups, or do you serve as a consultant in the biotech world at all?

GREENBERG: I've never had my own company. I have served as a consultant in a few instances. I've been involved as a witness or consultant involving patent disputes in biotechnology. I have participated in three significant cases in the biotech world.

ZIERLER: To give a sense of your graduate students and postdocs, what they go on to do, maybe as a pie chart, just in very rough numbers, who goes on to academia, who goes on to government and policy positions, who goes on to industry?

GREENBERG: Of my graduate students, about 15% have gone on to academic positions. Most have gone into industry, and almost all have stayed in science, with just one or two exceptions. A greater fraction of my postdocs have gone on to academic careers. I would say about a third of those, and the remainder have mostly gone into industry. Again, only one or two have left science and gone into, let's say, government regulation or consulting, which is an increasingly popular thing to do.

ZIERLER: Has your research gotten increasingly computational over the years?

GREENBERG: Not directly. We have been more involved in collaborations with computational chemists. In fact, we just published a paper a couple weeks ago in JACS where we were doing experiments on very large macromolecular molecules, nucleosomes, which are the monomer component of chromatin. We collaborated with a computational person that I know in France, who did molecular dynamics simulations of the processes that we've studied. Those are not the kinds of experiments that I could do on my own.

ZIERLER: Is this to say that your lab is not yet producing amounts of data where AI and machine learning is a valuable research tool?

GREENBERG: That is correct. The computational experiments that we've been involved with are more detailed calculations on individual molecules that are meant to help explain our experimental observations.

The Research Assets of Hopkins

ZIERLER: In what ways is having the hospital at Johns Hopkins an asset for you? What world does that open up for you in your lab?

GREENBERG: Well, not necessarily the hospital, but having the medical school and the faculty that are a part of that has been very helpful to me. I've had a number of productive collaborations. For instance, a few years after I arrived, we had a molecule that we were trying to test as a radio-sensitizing agent, and this is a great thing about Hopkins, I have found it to be a highly welcoming environment. I approached a person I had never met via email and asked if I could speak with them. This person was the head of radiation oncology at Hopkins. He welcomed me, and he had some of his lab members do experiments on our molecules because at that time, we couldn't do any cell biology.

His name is Ted DeWeese. He's now the interim dean of the medical school. He's just an amazing person and has been a great collaborator off and on, for close to 20 years. I've collaborated with other people at the med school. I currently have a joint NIH grant with my colleague Jim Stivers in pharmacology. Jim is an enzymologist who's an expert on DNA repair. We've collaborated a number of times. Hopkins in general, not just the medical school, is a really great place for me to be to interact with people and to learn.

ZIERLER: Being at Hopkins, being in the greater Washington D.C. area, is that an asset for you also, being in driving range of NIH, FDA, and the whole federal infrastructure?

GREENBERG: No, because when I submit a proposal, I can submit it by mail. [Laugh] There's really no advantage there. There is a great advantage in that D.C. has a lot to offer entertainment-wise and culture-wise. In terms of being close to NIH, I would say the greatest advantage was when I had my very first sabbatical. NIH has some laboratories in Baltimore. When people think of NIH, they think of Bethesda or maybe Frederick, Maryland, where NCI is. But the National Institute of Aging and the National Institute of Drug Abuse have research facilities in Baltimore. My very first sabbatical was basically an internship, if you will, in the Laboratory of Molecular Gerontology where I got own hands-on molecular biology and cell-biology experience so I could then come back here and try to do these kinds of experiments in my own lab.

Chemical Biology as an Experimental Breakthrough

ZIERLER: Let's move now to the main topic at hand. First, let's get terminology on the table. What does chemical biology mean to you, and how has that term shifted over the course of your career?

GREENBERG: I faced this question, interestingly enough, about the time I was moving to Hopkins. I was a graduate student with a phenomenal scholar named Jerome Berson, who coincidentally was the PhD advisor of Peter. I was in the process of moving from Colorado, and his wife, who was an incredibly impressive person as well, Bella Berson, asked me, "What is chemical biology?" This was at dinner at our house in Colorado. I've always thought of it in the following way, to distinguish it, in part, from biochemistry as well as biological chemistry. To me, biological chemistry and biochemistry are restricted in the molecules that they can work with based upon what nature will give them. But in chemical biology, we can use our ability as organic chemists or inorganic chemists to create molecules that nature can't create, that we can then use to study biological processes.

And so, how that has evolved, I think, is that when I was in Peter's lab, it was called bio-organic chemistry back in 1988 to 1990, and you had other people like Ron Breslow and Julius Rebek. What has changed over time is taking our ability to modify biological molecules and to use these molecules and to study processes in cells.

ZIERLER: The term bioorganic chemistry as sort of the forerunner to chemical biology, where is this simply a stylistic shift, and where does it really indicate new science, new techniques?

GREENBERG: I think the demarcation is the actual carrying out experiments in cells. And while you still, at the end of the day, think about it as a chemist, and what you analyze for might utilize some of the same methods, it's working with cells as opposed to working in the test tube.

ZIERLER: What aspects of this are a conceptual breakthrough, and what aspects are technological, where there are simply now instruments that are available to do this?

GREENBERG: I think overall, technology has helped all science, not just chemical biology. In the field of working with bio-macromolecules, I think mass spectrometry is a huge player technologically. But I also think it's a mindset, and it's the fact that it has become easier to do biological experiments. When I was a graduate student, cloning had basically first been developed. Making mutant proteins is something which is now kind of standard. I wouldn't reduce it to a kit, but it is a lot simpler than it was even when I was in Peter's lab. When I was in Peter's lab, even isolating a plasmid, which is a circular piece of single- or double-stranded DNA that you could then transfect into bacteria was nontrivial. Kits were first starting to come out back then. I remember working with one of Peter's graduate students, David Mendel, and we were testing some of these kits, and they didn't work very well. Now, everybody can get a kit, read the protocol, and the next day, having never done this, you can isolate plasmid DNA. I think for me as a chemist, biology has become more approachable due to it becoming more straightforward, more routine, and generally available. It's lowered the barrier for me.

ZIERLER: Of course, there are no solid demarcation lines, but the grand narrative in science from fundamental research to translational research, when did chemical biology start to offer clues anything how nature works that were relevant in improving human health, for example, drug discovery?

GREENBERG: Oh, I certainly think one example would be Stuart Schreiber's work in the late 80s and early 90s, working with rapamycin and the FKB binding proteins. I think that was probably a landmark example.

ZIERLER: What was so significant about Schreiber's work in this regard?

GREENBERG: Because he was able to utilize organic chemistry to identify binding partners and then make significant advances in terms of moving towards translational work, before the word translational was even really used. Stuart may not be the first, but I think of that as sort of the landmark example. Peter also eventually carried out his own cellular studies.

ZIERLER: This is more a sociology of science kind of question, but who is in the club of chemical biologists, and has that changed over the years, the kinds of people who would call themselves chemical biologists?

GREENBERG: It's funny because I'm not so sure people would call me a chemical biologist. [Laugh] Most of our work has been much more chemical. Of course Peter Dervan is one. Certainly, Carolyn Bertozzi, just a phenomenal example of this. And Pete Schultz. I would say those are two really excellent examples. Laura Kiessling is a great example. These are people who really have a deep understanding of chemistry and have just bridged the gap tremendously. Dennis Dougherty at Caltech, another example of a person who is a traditional physical organic chemist and has made tremendous contributions in chemical biology in the area of ion channels.

ZIERLER: You're mentioning people who come from a chemistry background who have gone into pursuing questions of biology. But has it become a two-way street? In other words, are there people coming from biology departments who recognize the power and tools of chemistry? Or is it still basically in that same mold?

GREENBERG: That's a tough one because I guess I don't know that group as well. This may just be the chemist in me speaking, but many people have said that it's easier to learn biology as a chemist than the opposite. And I actually think that at least in some ways, certainly in terms of synthetic chemistry, making molecules, that's true. What I often point out to my own group members is that when a chemist writes a paper, they write the experimental section. But when a biologist talks about procedures, they talk about protocols. And there's a big difference because in a protocol, you just follow instructions. And if it doesn't work, you kind of have to start all over, and the troubleshooting approach is a lot different. Whereas in synthetic chemistry, technique is really important, exactly how you do something with your own hands is important. I've had students come to my lab for rotations, and they view making molecules as, "Oh, that's just something I can do." The reality is, no, it doesn't work that way. [Laugh] I don't know of too many people who have gone from biology to chemistry. I'm having a hard time thinking of any, which could just be my own limitation. But certainly, chemists going in the other direction is very common.

Synthetic Lethality and Curing Disease

ZIERLER: Some of the big questions at the interface of chemistry and biology. When we think about curing cancer, the Human Genome Project, really broad, multigenerational, enormous scientific collaborations, what are the sensibilities, the technologies, the knowledge that either individuals or the subfield of chemical biology bring to these enormous projects?

GREENBERG: I think, for instance, the ideas of identifying particular pathways. I think of synthetic lethality, which is very much a molecularly oriented idea. Synthetic lethality is now a very popular way of trying to identify targets that would be attractive for treating cancer that rely upon identifying molecular shortcomings within a cell. If you have two genes in a cell. If either one is defective, the cell can survive. And often, the cell will survive having this one defective gene by relying more on another one with an overlapping function. But if both are defective, then the cell dies. It's very simple. Two genes producing products, one defective, the cell survives. Two defective, the cell dies.

Many cancer cells, because their genomic integrity has been damaged, are defective in a particular gene that might be important. One situation that's popular has to do with genes called BRCA1 and BRCA2. People who have a defective copy of one of these genes are more prone to developing reproductive cancers. The gene products are proteins that are transcription factors, and when they're defective, the cell has a deficiency in double-strand-break repair. That is why they're more prone to developing cancer. In cells that are deficient in double-strand-break repair, other proteins, such as poly(ADP-ribose) polymerase, PARP, help compensate. This is an enzyme that, in normal cells, if you knock it out or it's defective, the cell will survive.

But in these cancer cells that are deficient in double-strand-break repair because of their BRCA1 or BRCA2 defectiveness, knocking out or inhibiting PARP is cytotoxic. There's now a family of small-molecule drugs used to treat cancers selectively that target this kind of cancer cell. And this is attractive because the older way of treating cancer, using chemotherapy, you just give a poison that kills all cells, but it kills cancer cells more effectively because they're reproducing more rapidly. The desire to produce treatments that would be selective for cancer cells, synthetic lethality is such an example, and it really comes down to identifying molecules that overlap in terms of their function to exploit.

From NYU to Yale

ZIERLER: Let's go back and establish some personal history now. As an undergraduate, did you do a dual degree between NYU and Cooper Union, or was that sequential?

GREENBERG: It was a dual degree. It was a short-lived program that NYU had started the year I entered college.

ZIERLER: What was the nature of that agreement? Were you an NYU student, and Cooper Union had an attractive program? How did that work?

GREENBERG: [Laugh] That's complicated, a little funny story. NYU at one time had an engineering school, and they had two campuses, one in Greenwich Village, which still exists, and one in the borough of the Bronx. And for economic reasons, they had closed the campus in the Bronx before I got there, maybe in the 1960s or 70s, and with that went their engineering school. They no longer had an engineering school, and they wanted to have one again. Cooper Union is literally just a five-minute walk from NYU in Greenwich Village, it's on the Bowery. And for whatever reason, Cooper Union agreed to start this program where they would accept a small number of NYU students per year. My class was the first one, and there were four of us. We took all of our engineering classes at Cooper Union, and everything else was at NYU. By going to summer school, we were able to complete both degrees in four years.

ZIERLER: What was attractive to you about having the engineering component? Were you thinking specifically about chemistry and engineering, even chemical engineering?

GREENBERG: [Laugh] Yes, and that's the funny part of the story. This is really partly based upon my own naivete and ignorance. When I was applying to college, I was a native New Yorker, and I would be what you call nowadays an FLI, first-generation, lower-income. But back then, it was just, "My parents didn't go to college." I honestly didn't know much about what college would be like, and I thought maybe I'd be interested in engineering. I knew I liked science. I really wanted to go to Cooper Union, and this was again driven by my own lack of knowledge, because it was free. At first, I was on the waiting list at Cooper Union, then when NYU announced this program, I said, "Okay, I'll try that." I ultimately was accepted at Cooper Union, but my father gave me wise advice and said, "You should go to NYU, there will be more girls there." [Laugh] I went and did this joint program not really knowing what engineering would be like. I'm glad I didn't go to Cooper Union because I much prefer science over engineering, especially back then. Chemical engineering, you'd work in, let's say, a refinery or something. It's not like chemical engineering is today. It was very, very different, and it wasn't very appealing to me.

ZIERLER: As an undergraduate, did you have interests in biology? Were you even aware that there was this burgeoning interface between the two fields?

GREENBERG: I was unaware. There was a molecular biology course. I didn't take it, in part because I didn't have a lot of electives due to this program. Also, biology at NYU was this enormous class filled with students who wanted to go to medical school. It wasn't a very appealing class. In terms of research opportunities at NYU, the professor I did undergraduate research with had two subgroups that were totally separate. In one he was studying neurochemistry. I tried to get involved with that, but he pushed me towards his more traditional organic chemistry research. As an undergraduate, I didn't even have a course in biochemistry. Again, I didn't have the room to take such classes. While I was interested, I didn't really know anything.

ZIERLER: What do you think the value was of having that duality, the NYU and Cooper Union experiences?

GREENBERG: In hindsight, I probably would've been better served just going to NYU and perhaps taking a broader cross-section of courses. It probably wasn't one of my better decisions.

ZIERLER: You did okay anyway. [Laugh] Being a first-generation student, not having a good idea of what college was, you probably also didn't have a good idea of what graduate school was. Were there professors or labs that helped put you on that track so that a place like Yale would even seem in range?

GREENBERG: Absolutely. And yes, I didn't even really have a clear idea what a PhD was. When I was a sophomore taking organic chemistry, I said to one of my friends, "I really like it. Maybe I'll get a master's degree." The guy laughed and said, "No, no, you have to go get a PhD." Again, I've been very lucky throughout my life with having people who have, for whatever reason, looked out for me and taken me under their wing. At NYU, the first professor I had for organic chemistry is a man named David Schuster. He's retired now. He is a mechanistic organic photochemist, an organic chemist that studies photochemistry. David invited me to do research in his lab in my junior year. That was really very helpful, and he provided me with a lot of guidance. In fact, he helped me obtain my first exposure to biochemistry because in between the summer of my junior and senior years. I was able to get a summer position at Brookhaven National Laboratory, where I worked with a mechanistic enzymologist named Stan Seltzer. That was my first time even getting close to biological chemistry or biochemistry. But I had not taken a course in biochemistry, so how much I knew was questionable.

ZIERLER: Did Schuster have a connection at Yale or say this would be a great program for you?

GREENBERG: I'm not so sure you needed connections, but he provided a lot of guidance in terms of where to apply. Back then, it wasn't like now where when you are accepted, you get invited for a weekend of fun. In my instance, there were a couple places that weren't far away that, you could go and visit on your own. David certainly advised me to apply to Yale, and he gave me some of Jerry Berson's papers to try to read. He said, "This is a really great person." He advised me to apply to some other places as well. I applied to several places. I didn't get into all of them. I wasn't accepted at Harvard or MIT. And I thought I really wanted to go to MIT, but in hindsight, again, I think I was very lucky. Yale was a very good place for me, given my level of preparation. Actually, David advised me not to apply to Caltech, not because it wasn't an excellent place, but I think he knew that given where I was coming from and my level of preparation, I may not have succeeded at Caltech. Caltech was a place, at least when I was a postdoc there, where the grad students came in, and they already knew so much. I remember their candidacy exam, they would have to come up with truly original research proposals. I could not have done that as a second-year student. I needed a more structured environment, and Yale was a department at that time where you came in, and you took half a dozen courses your first year to get a foundation. I needed that, so it was the right place.

ZIERLER: Coming to Yale, did you know that Berson was going to be your advisor? Or that developed in real time?

GREENBERG: I did go visit there since it was so close to New York, and I met with him and a few other professors. For me, it was kind of love at first sight. He was a remarkable person, he was charming and interesting to speak with. I thought I wanted to work with him, but it didn't work that way. You didn't have an arrangement before you got there. After I got there, we still had to get to know each other and make sure it was the right fit.

NMR and Organic Molecules

ZIERLER: Tell me about Berson's research. What was he known for?

GREENBERG: By the time I worked with him, he was already in his 50s. He was very well-known for working on things like thermal rearrangements and the testing of the rules of orbital symmetry controlled reactions. These were the Woodward-Hoffmann rules, which Hoffmann and Fukui shared the Nobel Prize for. He also was very well-known for molecular rearrangements in general, carbocations and things like this. The area that I worked in was one that was an outgrowth of his interest in trying to push the limits of orbital symmetry controlled reactions, the study of diradical molecules. By the time I joined his group, he had a project with several people working in it where the idea was to study biradicals that were pi conjugated, and due to their structure, they could either have singlet or triplet ground states.

The idea of having a singlet ground-state biradical is unusual, and only very special molecular connectivity would lend itself to that. This turned out to be an area that was of growing interest in the 80s and 90s, and Dennis Dougherty was involved in this as well, where people were trying to make ferromagnets. And there still are some people trying to do this, where they make polymers of radicals, with the intent that the spins would communicate with one another to make high-spin molecules. In a sense, Jerry Berson was working on the very foundation of this, what kind of molecular connectivity would lend itself to high-spin or low-spin. The other area he was still working in was molecular rearrangements and pushing the limits of stereoelectronic control.

ZIERLER: With all of this work, how did you develop your thesis topic?

GREENBERG: You didn't develop it on your own, you met with him and were presented with a menu of potential projects. I originally chose one in the biradical area, in part because I liked the idea that it would expose me to a variety of types of experiments. I would synthesize molecules and study their reactivity. We also would use spectroscopic methods to characterize these reactive species. I was very attracted to the idea of having a multiplex approach and learning a variety of skillsets. My original project didn't work. We couldn't make the molecule.

I ultimately got very lucky, I joined a project, also a biradical project, where the molecules were the first ever ground-state singlet biradicals. I was then even luckier that at that time, there was a junior professor who had just joined Yale named Kurt Zilm, who's still there. He's a solid-state NMR spectroscopist, and he wanted to develop low-temperature solid-state NMR using magic-angle spinning so you could observe reactive intermediates. I was in the right place at the right time. With Zilm, we did the first NMR of a biradical at low temperature. Because it's a singlet, you could readily detect it by NMR. That was the spectroscopic proof that these molecules were ground-state singlets. At the same time, it was the first example of characterizing a reactive intermediate by solid-state NMR. I was very, very lucky.

ZIERLER: What aspects of this were significant from a fundamental research perspective, and what aspects were what we might call technology proof-of-concept, that these technologies were really valuable in this area?

GREENBERG: The fundamental chemistry is, concerned with how the structure of a molecule, its connectivity, determines whether or not Hund's rule is violated. Basically, higher spin is typically more favorable energetically than lower spin. But could the structure of a molecule control whether a diradical should be a triplet or a singlet? As I said, this provides the foundation for the design of high-spin molecules that people were interested in. As far as the technology goes, I didn't really develop that, I was just lucky to collaborate with Kurt Zilm. It was the idea of being able to spectroscopically observe reactive species. These are molecules that, in solution, would exist for a microsecond. Now, you can actually take an NMR just like you would of a normal organic molecule. That was really Kurt Zilm's doing.

ZIERLER: At Yale, were you beginning to think about biology at all? Was Berson's lab thinking about biology at all?

GREENBERG: No. Berson's lab wasn't. I was told by a senior graduate student that about five years before I got there, he was. He was trying to use biradicals as probes in enzyme binding sites. But my understanding is that it didn't work out. At that time, Yale didn't really have any bio-organic chemistry. It had a large organic group, but it had four classical physical organic chemists and four synthetic organic chemists, one of whom was Stuart Schreiber. But when I first started graduate school, he was solely doing synthesis. There was bio-physical chemistry. Don Crothers, Jim Prestegard and Peter Moore. Gary Brudvig came while I was there. I didn't have any experience in bio-organic chemistry. And as an undergraduate, I didn't have biochemistry coursework experience. At Yale, I audited biochemistry at the undergraduate level one semester because I was interested and wanted to learn. However, I mostly tried to learn by reading the literature. Back then, I had my own personal copy of JACS, and that's where I would read the hottest and best stuff. That was how I was able to identify bio-organic chemistry as something that I was interested in.

ZIERLER: As you were thinking about potential career paths, was biotechnology an option? Were people talking about biotech in those days, or that's still too far afield?

GREENBERG: Not at Yale, not in my peer group. People weren't really talking about biotechnology. By the time I graduated, it was the mid to late 1980s, so this certainly existed. But I wouldn't say that people were discussing it at that time at Yale.

ZIERLER: You were firmly on an academic path. The next stop for you would be postdoc.

GREENBERG: Next stop was going to be a postdoc, but I wouldn't say I was definitely on an academic path. I wasn't sure I could be an academic, and I wasn't sure, given life's other issues, whether that would be possible. Having a potential spouse, things like that. While being in Berson's group inspired me to want to be an academic, I didn't know if I would succeed.

Joining the Dervan Lab

ZIERLER: When it was time to think about postdocs, how did Caltech get on your radar? Did you hear about Peter Dervan at that point?

GREENBERG: I got interested in biological chemistry or bio-organic chemistry by reading the literature. I knew that I wanted to continue doing mechanistic chemistry. I love solving puzzles, and to me, mechanistic chemistry is like solving puzzles. I knew that as a graduate student, as I was getting closer to graduating, I was thinking, "If I could be an academic, I'll have to identify my own area of expertise. I would love to be able to study how biological molecules react." I learned of Peter's work by reading the literature, and by the mid-1980s, Peter was really prolific. He had already hit his stride, and he was the hottest thing. I gave a group meeting on Peter's research. Again, to Jerry's lab, I had to start with the structure of duplex DNA because my group members wouldn't necessarily know. I had to learn about it. [Laugh] This was not something I learned as an undergraduate.

When it came time to apply for a postdoc, Jerry Berson was not a heavy-handed person, so he wouldn't tell me who to apply to. I went in with a list of people, and he actually was opinionated, and he commented on some of the people like, "Well…" so I took that as a negative. [Laugh] But I ended up applying to Peter and a couple of other people. And when I got the offer from Peter, in those days, you got letters. I excitedly went down to Berson's office, and he said, "That's the one you want." For him, that was really a strong opinion. And I agree, it was. And I don't regret it one bit. It was really a great opportunity.

ZIERLER: If you can remember the details of what you were trying to convey at that group meeting, in just reading the literature, what Peter was doing, what was so new? What was so earth-shaking about what he was doing in the mid-1980s?

GREENBERG: Back then, a popular term was molecular recognition. The idea of making molecules that would bind non-covalently. Of course, the idea behind this is to try to mimic and understand, for instance, how molecules would bind to DNA or how proteins would bind to small molecules. Number one, how most organic chemists were doing this–well, all except for Peter at that time–was, they were doing it in chloroform, where the molecules they create don't have to compete with water for hydrogen bonding. They weren't studying the biological molecules under relevant conditions.

To help understand non-covalent binding, Peter developed chemical footprinting, which enabled people to determine where molecules were binding on DNA. That's important in terms of identifying sequence selectivity. You have a small molecule that binds to DNA. Where? What sequence does it prefer? Peter developed chemical tools that allowed you to do this quantitatively. He also developed affinity cleavage, which was the complement of footprinting. If you think of photography, one would be the negative, one would be the positive. In footprinting, you cut the DNA everywhere except where the molecule binds. In affinity cleavage, you cut the DNA only where the molecule binds.

Peter was designing molecules that recognize and bind non-covalently to DNA in water. The molecules could be used as synthetic nucleases, molecules that would cut DNA at a particular DNA sequence. You could imagine why being able to identify a single sequence in DNA would be important, both therapeutically and with respect to the Human Genome Project. It was tremendously exciting from the standpoint of an organic chemist actually studying biological molecules in water. Now, it seems so simple in hindsight. Once people know how to do things, it's always simple. It was phenomenal at the time.

ZIERLER: The misgivings coming from NYU that perhaps you lacked the preparation to go to Caltech for grad school, did that translate at all? Berson was very clear that this was the spot for you. Did those concerns at all translate to your postdoc, or you had resolved all that and felt ready for Caltech?

GREENBERG: I think in hindsight, I was probably not as strong a scientist I had thought I was. I wasn't worried about going to Caltech the way I would've been as a graduate student, but when I arrived in Peter's lab, I felt like I had gone up another level. The people in Peter's lab, the people in other labs that I met at Caltech, it was definitely a higher-energy environment than I'd come from at Yale, and I had the feeling that people were working on more urgent cutting-edge problems. Certainly in Peter's Lab.

ZIERLER: As a New Yorker, what was arriving to Pasadena like for you? What were your impressions?

GREENBERG: It was really nice. It's funny you should say that because as a New Yorker when I was young, I always felt so worldly because I grew up in New York. And I realize now how silly that was. Pasadena was convenient, it was easy to live there, it made doing research easier because you didn't have any hassles, you didn't have to get on a subway like I did as an undergraduate to go to school. I lived five minutes from the lab. You didn't have to deal with snow or rain in the winter. It was great. Pasadena was a very comfortable town. I didn't have a whole lot of time for entertainment, but traffic wasn't nearly as bad in 1988 as it is now. I would get up early on a Sunday morning and run down to the beach for a couple of hours. I used to play softball on Sundays for a long time with a group of people I'd met through Alanna Schepartz,. [Laugh] I used to go and play Sunday morning, then go back to Pasadena. Again, it was a really nice, comfortable place.

ZIERLER: From reading Peter's work in the mid-1980s, when you joined in '88, '89, was he still on that same trajectory, or had he moved on to new projects at that point?

GREENBERG: He was in the same general area, and he was soaring. Because by the time I arrived in the late 80s, the Human Genome Project began in 1990. Peter had published, I think in 1987, the first triplex paper, triple-helical DNA. Triple-helical DNA was quite the splash because conceptually one could synthesize a single oligonucleotide, potentially on a machine, that could recognize and potentially cut the human genome at a single place. It was incredibly timely that Peter had gotten involved in triplex DNA. I couldn't speak to what Peter knew and thought of and how he arrived at this.

But in terms of his trajectory, he was just soaring. And triple-helical DNA, at that time, there were several people in the lab working on it, including Eric Kool. Peter had not yet moved into doing cell biology in his own lab, but he was continuing along the path of developing a molecular code, if you will, to recognize any sequence of DNA. Interestingly, as I mentioned, the triple helix was really, really hot. But by the time I left in 1990, the small molecules that bind in the minor groove of DNA, the polyamides that he had started with, were coming back in favor because people had determined structurally, and Peter, also had figured this out from his own binding studies, that these small molecules could bind as dimers. Two of them would stack and bind in the minor groove.

Peter recognized that this was a way to increase and enhance the ability to recognize specific sequences because one molecule would recognize the bases on one strand, and the other one would recognize the bases on the opposite strand. The student who turned this project around and made the minor-groove-binding molecules extremely important was Milan Mrksich. Milan's now at Northwestern. Peter's trajectory was soaring, and research continued along within the general idea of, "Can I design molecules that would recognize DNA sequence specifically?" The progression was minor-groove molecules, then triplex came up, minor groove was going down, but then minor groove came back up, and he ultimately did develop a code to recognize all four possible base pairs in the minor groove, utilizing this idea of two separate small molecules that were covalently linked, bispolyamides.

ZIERLER: Were the students and the postdocs all working in this one area? How dispersed was the lab? What were the different things it worked on?

GREENBERG: By the time I was in the lab, there was one student, Jim Hanson, who was working on electron transfer. A popular physical organic chemistry problem in the early to mid 1980s was the so-called Marcus– inverted region, an idea developed by Rudy Marcus, another Caltech faculty member. The idea is that in the Marcus-inverted region as reactions become thermodynamically more favorable, they react more slowly. A number of people, including Peter, had been designing molecules to test this idea. Other than Jim, pretty much everybody–the group was around 20 people–was working on some aspect of the molecular recognition of nucleic acids, whether it be using small molecules, the polyamides, DNA binding proteins, or triple helix.

ZIERLER: What was Peter's style? Was he in the lab all the time? How accessible was he?

GREENBERG: Peter traveled a lot at that time. But when he was around, Peter was very interactive. I remember being in the lab on a Saturday morning when I first started, and Peter came down looking for coffee. [Laugh] When you have a group of 20 people or more, and you have all the world knocking on your door to ask you to do this and that, Peter's time was limited. But when you were with Peter, it was really quite invigorating. He is just so dynamic, and he was so excited about what he was doing. He conveyed such enthusiasm that you would meet with him, and maybe you didn't have very good results, but you would leave feeling like, "Okay, I can do this," and you'd run back to the lab. He certainly had that effect on me. And my projects didn't work fabulously in his lab, so I needed that inspiration. [Laugh]

Excitement in the Laboratory

ZIERLER: The energy of his lab, the sense that he's soaring at this time, what did that teach you more generally about how to be successful in science?

GREENBERG: Well, my experience at Caltech was valuable in many ways. Of course, I'd like to think that I convey the same kind of excitement about what I do even 30 years later. I'm still very, very excited about working with my group and trying to do new research. Peter also taught me some practical things. He told me, "You don't ever want to be limited in what you do by physical resources. Always make sure that your students have what they need to get the job done," so I've always been very cognizant of that. He also gave me some very good parting advice, given what he knew my research goals were going to be at the beginning. They were very much a combination of what I did as a graduate student and as a postdoc, where I was going to merge physical organic chemistry with studying nucleic acids. He said, "You're not going to be able to look at these large molecules with the same detail always. There are going to be some things you're just going to have to let go of." And that was very helpful advice. But Caltech was a great place to prepare to be an academic.

ZIERLER: What were the difficulties in your experiments? What was so difficult?

GREENBERG: After a few months of working on a project where we were trying to create a modified protein to sequence-specifically bind and cut DNA, Peter encouraged me to switch projects. He felt that as a mechanistic chemist coming from Jerry's lab, I would be well suited to understand how the complex Peter was usually using to cut DNA, which is referred to as iron EDTA, cleaved DNA. This turned out to be a really complicated problem. [Laugh] In the end, we did learn quite a bit, but as I delved into it, I realized, "This is something people have been working on a really long time. This is not necessarily something I can get all the answers to in a couple of years as one person." It was a challenging project. But we did do some experiments that were really good, we just didn't publish them. Ultimately, someone named Tom Tullius did essentially publish part of what I had done with Peter's lab about eight years later in PNAS. [Laugh]

ZIERLER: Was the term or the field of biotechnology more real to you as a postdoc at Caltech?

GREENBERG: Absolutely. Because by that time, you already had companies like Applied Biosystems that were making sequencers and things like this. And I believe Gilead existed at that point. Certainly, ISIS existed. Being at Caltech in California and being in that environment, you knew of these companies.

ZIERLER: Did you think about industry at that point?

GREENBERG: I did. In fact, I interviewed for both. I was engaged at the time to someone I met in graduate school. She's my wife now, Fran. We've been married 33 years. I didn't know if I could get an academic position where Fran could also pursue her career. She had graduated from Yale about a year after me and went directly to Merck in medicinal chemistry. Which, as a synthetic organic chemist, which is what she was trained as, that's a great job. When it came time for me to look for a position, we sat down with a map to see, "Okay, these are where academic posts are this year. Do we think there are companies?" Because the internet didn't really exist yet. I applied to some places, and I also interviewed with companies, some of which recruited at Caltech.

We decided, "Okay, we'll see how this works out." I ultimately did get a couple of industrial offers. One was at Merck, one was at Union Carbide, which doesn't exist anymore. I had two academic offers. One was at Colorado State, where I went, and the other one I couldn't seriously consider because there was really no job in the area for Fran at that time. It was pretty interesting because one of the senior faculty called me and said, "Are you going to come?" I said, "I'm very interested, but it doesn't look like Fran can find a position." This was in Atlanta at Emory. At that time, there wasn't a lot of biotech down there. There were only things like Georgia Pacific, where one would make toilet paper or something like that. And that just wasn't fair to try to ask Fran to do something like that.

Then, when I explained this to the person, they said, "Well, just have your girlfriend come down anyway, and she'll find something." And I said, "No, I'm sorry, I don't think that's going to work. She has a PhD from Yale just like I do." [Laugh] I couldn't go there. But fortunately, one of the senior faculty at CSU, Al Meyers, who passed away more than 15 years ago, was very, very active in the local Colorado scene. He helped us identify some biotech companies. And Fran gave up her career at Merck in medicinal chemistry to go to one of these biotech companies, which didn't pan out. But then, she ultimately moved to what became Ribozyme Pharmaceuticals, which was a company in Boulder that Tom Cech had started to turn catalytic RNA into a drug, and that turned out to be a really good opportunity for her. We both compromised, and we've done so ever since.

Faculty Appointment at Colorado State University

ZIERLER: The transition point from postdoc to faculty member. Chemical biology, was that just starting to come into play, or this still takes some time?

GREENBERG: It was starting to come into play by that time, by around 1990.

ZIERLER: And you would've called yourself a chemical biologist? Would you have fit that niche in an academic department?

GREENBERG: I did not at that time because the research I was doing was completely in the test tube. I was in a funny spot because I was doing what I would call bio-organic chemistry, and yet chemical biology was becoming the term. Chemical biologists could look at me and say, "He's not a chemical biologist," and that would be fair. But physical organic chemists would look at me and say, "Oh, he's working with DNA. He's not a physical organic chemist." I kind of fell between the cracks. [Laugh] And definitely, our program lagged behind in terms of embracing true chemical biology. Now, I would say that definitely some projects in our lab are chemical biology projects.

ZIERLER: As an assistant professor, tell me about starting up your lab. What was most important for you?

GREENBERG: To prove that I can do independent science and that what I was working on was something people and funding agencies would care about, something that would attract students, and that I would be able to train students to be good scientists. For me, the latter has always been really important. And when you start at a place like CSU, which is a lot different than Caltech, the personal training of students is really important. It's integral to the job. I've always been proud of the fact that I've given my students a lot of hands-on training.

ZIERLER: What would you say was the research or impact that ultimately gained you tenure?

GREENBERG: It was one particular project, which evolved over many years. It was the idea of trying to study how DNA is damaged, so this is the connection to Peter's lab. We approached it using organic chemistry to synthesize nucleosides that we would then incorporate into synthetic DNA using solid-phase oligonucleotide synthesis. And when photolyzed these molecules, produce a particular radical. This radical would be of interest because it's a radical that might be produced by, an anti-tumor agent or radiation. But because we were using photochemistry on a homogeneous synthetic oligonucleotide, we could study the reactivity of that molecule in greater detail and clarity. As an assistant professor, I contemporaneously developed this experimental approach–I didn't know I was going to have competition [Laugh]–with a very prominent chemist, Bernd Giese. You may have heard of him as one of the competitors with Jackie Barton in electron transfer. I was an assistant professor at CSU competing with Giese, who had his own mass spectrometer, and we didn't even have a mass spectrometer at CSU that I could use for this project. However, we were able to contribute to the field and that was the project that got me tenure.

ZIERLER: Being in Colorado, relatively remote from the biotech hubs of New York, San Francisco, and San Diego, did you feel like you could contribute? Or that simply wasn't possible?

GREENBERG: I could. CSU is in Fort Collins, but for a town its size, Boulder, which is an hour away had a fair amount of biotech. Amgen originally had a presence there because of Marv Caruthers. And there were other companies there. Ribozyme Pharmaceuticals sprang up. Larry Gold started what was originally called NeXagen that became Nexstar, that was using aptamers as potential therapeutics. They and Ribozyme, I believe, were in the same business park in Boulder.There even was a sort of a biotech society that provided small seed funding to faculty in the area. I got to know people like Larry Gold a little bit at the time, and certainly the people at Ribozyme. And then, a company called Array BioPharma started, because Amgen increased its footprint in Boulder and then abruptly ended it. They had hired a large number of medicinal chemists from big pharma who came to Colorado and loved it. When Amgen closed down its site, they started Array. Array went on for many years and was bought a handful of years ago by Pfizer, I think. There's always been a reasonable amount of biotech in the Boulder area stretching to Denver. Sure, nothing like California, but more than Baltimore.

ZIERLER: Over the 1990s, as the Human Genome Project was really picking up speed, was that relevant for you at all?

GREENBERG: Absolutely. Because one of my other projects was a synthetic project concerned with synthesizing call bioconjugates of oligonucleotides. We had an NSF grant for several years to synthesize oligonucleotide bioconjugates. This project helped some of my students get jobs in this area as well.

ZIERLER: Tell me the decision-making about transferring to Hopkins.

GREENBERG: It was both personal and intellectual. The Chemistry Department at CSU, particularly in synthetic organic chemistry, was really quite good. Once again, I was kind of a loner in the way that I wasn't a synthetic organic chemist, and CSU did not have a strong bio-organic or chemical biology group. Also, the university as a whole wasn't very strong outside of chemistry. Within chemistry, Al Meyers became a member of the National Academy of Sciences, and we had other people like Lou Hegedus and Bob Williams. These were really successful people who brought in a lot of graduate students. But if I was going to grow intellectually, it was going to be hard there.

There was no medical school there, and as I mentioned, the departments outside of chemistry–biochemistry was separate–were not particularly strong. From the intellectual standpoint, I viewed Hopkins as a place where I could grow. Because as I mentioned, there's just so much chemistry here at the biological interface, throughout the university, not just the medical school. I've had so many excellent interactions. For example, our work in nucleosomes probably would not have been possible if I'd stayed at CSU because here at Hopkins, I have a colleague in biophysics who was willing to take one of my graduate students into his lab for a short while and let him learn how to handle these molecules, and bring that technology back to us.

Chemistry and Biology Interfaces across Hopkins

ZIERLER: To clarify, circa 2002 when you joined Hopkins, this interface between chemistry and biology, that was already in train, already happening at Hopkins?

GREENBERG: Oh, yes. Here, it was organic. It was evident in people's research throughout the university. When I came here, for instance, in the pharmacology department, Phil Cole, who had been an undergraduate at Yale when I was a graduate student, had gotten his MD/PhD here and had gone on and started his own academic career at Rockefeller. Phil had come back to Hopkins to be the head of pharmacology. He hired Jun Liu, who is still here. Jun was in Schreiber's group, and was involved in the FK binding protein, rapamycin story. Phil hired Jim Stivers, who I mentioned, a mechanistic enzymologist. They had a number of chemical biologists in their department, and these are people who could easily be in a chemistry department.

And that is the case throughout. For instance, biology, which is next door to chemistry at Hopkins, has historically had a very strong biochemistry group. When I arrived, there was a professor named Maurice Bessman, who passed a few years ago. Maurice was working on DNA repair enzymes. There was a natural overlap for me. There are two biophysical departments here at Hopkins, one on the college campus, and one at the med school. In the biophysics department at the college campus, Sarah Woodson who works on RNA folding was here and still is. Sarah and I have worked together. There were just so many people here I could naturally interact with.

ZIERLER: Given all of this interface, the simplicity of the names of your key departmental appointments, Department of Chemistry, Department of Biology, presumably, these are the same names they had maybe 100 years ago. Has there ever been any discussion about changing those names to be inclusive of this interface? Having a biochemistry, or chemical biology, something that was indicative of all of these interactions?

GREENBERG: The only department that's changed its name since I've been here is the Chemical Engineering Department, which is now the Department of Biological and Chemical Engineering. I think this is very typical of what really is going on in chemical engineering. As I mentioned earlier, chemical engineering is so different now from when I was an undergraduate. People in chemical engineering here do protein engineering. They're not working on refinery projects. To my knowledge, there haven't been any discussions about changing names necessarily. But what you do have at Hopkins are a lot of umbrella programs. These are graduate programs that are often funded, at least in part, by an NIH training grant. There are many NIH training grants at Hopkins. I was the founding director of the Chemical Biology Interface program here. We have an NIH CBI training grant. It's now in its fourth term. Steve Rokita, my colleague, took it over after me. In addition, we have cellular, molecular, developmental biology, and we have a program in molecular biophysics. All of these programs have overlapping faculty. While the department names haven't changed, we have cross-fertilization through our graduate programs.

Entering the World of Cancer Research

ZIERLER: How did you get more involved in cancer research at Hopkins?

GREENBERG: That was part of my motivation to try to grow intellectually and realizing that just studying reaction mechanisms in the test tube doesn't appeal to an enormous number of people. It was also partly to try to convince people, that the type of fundamental studies that we're carrying out are things that we all should care about. And what better way to prove that than to take a fundamental discovery and say, "Ah, this could be useful for solving this problem"? When you're focusing on things like DNA damage, cancer is just a natural connection since so many treatments involve damaging DNA. And of course, modifying DNA is an underlying source of cancer.

ZIERLER: How fundamental to the existential questions of cancer research, how cancer develops, how to cure cancer, is the mystery of DNA damage to those overall questions?

GREENBERG: I think it's a fundamental question both in the formation and the treatment of cancer. For instance, there are forms of DNA damage that are referred to as biomarkers. There's one particular modification of one of the four nucleotides, deoxyguanosine, called 8-oxo-G. It's a chemical modification. This is used as a biomarker for prognosticating the development of disease and cancer. In terms of the treatments, there are lots of examples, and they tie in with the development of cancer as well. Because when your DNA is damaged, you also have repair. Faulty or inept repair can ultimately give rise to cancer by not fixing mutations. Then again, DNA repair can also thwart potential therapeutics that target cancer by damaging DNA. The fundamental aspects of DNA damage, DNA repair, the development of the disease, and the potential treatment are all really interconnected.

ZIERLER: In developing the Chemical Biology Interface program, having sort of an administrative formalism to all of these interests and ideas, what was impactful about that that you would not have been able to accomplish simply by pursuing your own research and the research in your group?

GREENBERG: I would say there, it's mostly educational. It's attracting students who have a particular interest, where they might not have come to Hopkins because they couldn't get the exact education that they want. This is where Hopkins not being a very large place–the chemistry department is only 20 or 21 faculty right now, is at a disadvantage. By having umbrella programs, you can attract students that have inerests that are broader than the department can accommodate. I think it's really impactful from a graduate education standpoint. The CBI program at Hopkins is a degree-granting program. In a lot of training programs the student receives a PhD from whatever the home department is. We created a PhD in chemical biology, and a curriculum that is distinct for the students in this program. Then, of course, there are the research opportunities they can choose from. If they had come to Hopkins Chemistry, they'd have, three or four faculty they might be interested in, but if they come as a CBI student, there are now 30 faculty to choose from.

ZIERLER: I wonder what can be read into this in terms of the overall maturation of chemical biology. In other words, thinking back to when you were a young faculty member at CSU, and maybe people didn't know what to do with you because you weren't quite this, and you weren't quite that. By the time you had established CBI, is part of the broader message here that this is a mature discipline, there are graduate students who are interested, and there needs to be an educational infrastructure that responds to that interest?

GREENBERG: Absolutely. I don't think we're unique in that regard, but absolutely.

ZIERLER: Has CBI gone on after you stepped down as director?

GREENBERG: Yes, it still exists. My colleague, Steve Rokita in chemistry, has been directing it for 10 years. It's existed for almost 20 years.

ZIERLER: Your affiliation with the department of biology and becoming a member of the Kimmel Cancer Center, I would've thought that this might've come earlier, that these would've been affiliations for you to have right at the beginning. What explains that relatively late transition?

GREENBERG: To be honest, they're largely just courtesy appointments. The biology appointment is by being part of the training program. I don't teach in biology, I'm just part of their umbrella graduate program, and as a result, a member of their department. The Kimmel appointment, I don't really know how that came about. I think it was due to my collaborations with people in the School of Medicine. But again, they're largely courtesy appointments. The Kimmel appointment, opened up access to some technological resources. But I wouldn't say it's a transformative appointment in terms of what we do, and it doesn't make it any different in terms of collaborating with people.

ZIERLER: What about the department of oncology? Is that in a different category?

GREENBERG: No, that's largely the same. It's all part of the Kimmel appointment.

ZIERLER: Moving our conversation closer to the present, when COVID hit, did you have any access points to COVID research? Is there any relevance in virology to the kinds of things that you do?

GREENBERG: No, there wasn't for me. We were shut down because the only way you could do research is if you were working on COVID related problems.

ZIERLER: How was that transition for your lab? What were you able to automate? Where was it about just reading from home? How did you and your lab manage during that time?

GREENBERG: I was on sabbatical, but I spent most of my time trying to keep my group engaged. We continued to have group meetings, where we would discuss journal articles. To try to make their time productive, and to help them think about their projects, I had the students write introductions and background sections for their theses. It created a lot of work for me because I had to read and edit all of them. [Laugh] But that's okay. It was difficult for students. My last student from that era is still here, but she's going to graduate in about a month. We were shut down for three months, then for four months, we were on part-time, partial-access. Our research really took a hit.

ZIERLER: Bringing the conversation right up to today, what are some of the big areas your group is now pursuing?

GREENBERG: I'm actually really excited, we're about to write up a paper with the final student from the COVID era. It relates to the fact that so much of what we do is fundamental science, and the question is, how can I make that interesting to, let's say, a person at a cocktail party? We published a paper, almost five years ago, in PNAS, where we discovered that the major form of DNA damage produced by common alkylating agents, involving the simple addition of methyl group to a nucleotide, reacts within chromatin to produce DNA protein crosslinks. DNA proteins crosslinks are a more deleterious form of DNA damage. The initially formed methylation product, which biologists have said was unimportant, is produced by molecules such as temozolomide, a drug used to treat glioblastoma.

In the PNAS paper, we also showed that this chemistry occurs in the cell. For me, discovering fundamental chemistry and showing that it happens in a cell when using agents that are used to treat cancer, is very exciting. What we're about to publish, which we didn't show then, is, "Okay, this happens in a cell, but is it important biologically?" We have evidence that this chemistry not only happens in the cell, but it contributes to the cytotoxicity of agents that produce the initial alkylation damage.

And it turns out that someone had observed in 1973 that DNA protein crosslinks are formed from these products, but they disavowed it in the paper they published in Cancer Research. They basically said, "It can't be a DNA protein crosslink because these molecules can't produce this." This makes me take a step back and think, "People have been using these kinds of molecules for 70 years to treat cancer." If people had not thrown that result out 50 years ago, would it have changed how cancer cell biologists think about how these molecules work? And would it have changed how they pursued their own research and how they treat cancer using these molecules? To me, this is very intriguing. We are applying what we've discovered in the test tube, that happens in the cell, and is physiologically significant to make new molecules that will selectively produce DNA protein crosslinks in higher yields. At a minimum, such molecules will be useful tools for studying the repair of DNA protein crosslinks in cells, and at the maximum, well you can dream as big as you want.

ZIERLER: What exactly did the researchers recant in the 1970s, and what did they get wrong that they shouldn't have recanted?

GREENBERG: They didn't recant it, they just said that their observation couldn't be attributed to DNA protein crosslinks formed from alkylated DNA. These people had done a series of experiments, where they would treat cells with various alkylating agents. Some of them were simple alkylating agents, so-called monofunctional ones, and others were things like nitrogen mustards. Nitrogen mustards have two reactive components, and readily form DNA protein crosslinks. After treating cells with these agents, they would lyse them, and they would do a phenol extraction. When you do a phenol extraction, DNA goes into water, proteins go into phenol, and DNA protein crosslinks appear at the interface. These people observed this, and they referred to the material at the interface as a DNA protein complexation. They explicitly wrote, "It can't be DNA protein crosslinks because monofunctional alkylating agents produce them, too." [Laugh] That's what they wrote in their paper.

The Relevance of Fundamental Chemistry

ZIERLER: This could be a game-changer.

GREENBERG: I don't want to dream that big, but for me as a scientist, it is exciting. It's exciting because we've shown that fundamental chemistry has direct relevance to something that many people have studied in terms of its biological consequences. I don't want to over hype it. I wouldn't say it's a game-changer, particularly when one refers to cancer because it's such a complicated disease. But it's certainly, I think, a significant increase in our understanding. And now, it provides us with direction for how to try to design molecules that might actually be more beneficial, more useful in a cell.

ZIERLER: For the last part of our talk, I'd like to ask a few retrospective questions about your career, then we can end looking to the future. First, what has stayed with you from your Caltech postdoc experience? What did you learn from that that has really been close to how you've developed as a professor, how you've developed important research?

GREENBERG: It's partly both how Peter ran his lab and how he was able to inspire people, that personal interaction. It was also the high caliber of scientists I was surrounded by. And Peter set the tone here. He had really high standards for how experiments should be and how good data should be. And of course, the identification of really important problems. That, I think, is more limited by my own intellectual power. I wish I was as visionary as Peter in my choice of problems because clearly, he is truly visionary in that way. But it's definitely an appreciation of just what good science is and what the quality of the end product should be.

ZIERLER: In your own research trajectory, the delineations between fundamental or basic science and translational science, have those become more blurred? Will chemical biology or the kinds of questions you're looking at not, at some point, will it not be very relevant to think about it in terms of one or the other?

GREENBERG: I don't think it will go from one to the other. It's going to vary based upon the individual. Over my entire career, there have always been people who have focused on problems that are more applied. Combinatorial chemistry was a great example of that. People didn't necessarily develop new science, but they developed methods that provided molecules that would be potentially beneficial. And that's fine. I think those are important problems that people will continue to work on. But at the same time, people have continued to make discoveries of fundamental importance that are very exciting.

In nucleic acids, a beautiful example of this is, back around 2009, there were five nucleotides. A, G, C, T, and 5-methyl-C. And within three years, there were three more. The oxidation products of 5-methyl-C. Those discoveries launched a whole field of epigenetic-related search, focused on so-called epigenetic bases. This is an example of a very fundamental problem, which has potential applications translationally. I am confident that there are exciting problems of fundamental interest that remain to be discovered. And then, of course, the two go hand-in-hand. When you discover something that's fundamentally new, it potentially opens up translational problems. I don't think fundamental science will go away. There's still so much left to understand.

ZIERLER: As you've so nicely conveyed, you're clearly a beneficiary of excellent mentorship all the way from undergrad through postdoc. What are the big takeaways, and how have you tried to pay that forward in your own capacity as a professor and mentor?

GREENBERG: I guess maybe you should ask my students that. [Laugh] But I'm very proud of the fact that I have always tried to help my students to become complete scientists. I have always spent a great deal of energy helping them learn how to write about science and how to give talks. This is particularly important for my many international students. I've also always tried to encourage my students to explore their own interests. How I do that, for instance, is, in group meeting, to this day, we don't just talk about our research. We talk about the literature. Students, once a year, give a literature seminar, where they pick a topic. And I tell them, "I don't want it to be anything related to our research. I want it to be what you're interested in so that you can identify problems that you might want to work on or an area that you might want to be a postdoc in." And I participate by giving presentations, because I want to lead by example. I take the time to present journal updates and present papers from the literature. In fact, I'm getting ready in two weeks to present Eric Kool's most recent paper from Nature Chemistry, which I think is just a beautiful, innovative paper.

ZIERLER: The Dervan group lives on.

GREENBERG: Yes. I've been very fortunate to not only have great mentors, but I've overlapped with so many tremendous scientists, particularly when I was in Peter's lab, of which Eric Kool is one.

ZIERLER: Finally, last question, looking to the future. As you indicated, you don't want to express hype because cancer is such a difficult and complex human challenge. Best-case scenario, if you can dream big, how will this research contribute to those overall challenges?

GREENBERG: Well, best-case scenario, the molecules I alluded to just a few moments ago will be effective in cells. And who knows? Maybe they would be therapeutically useful. And in our other area we've been working on, polymerase inhibitors, I mentioned to you the idea of synthetic lethality. We demonstrated about two, three years ago that the target we're working on is also synthetic lethal with BRCA1 cells. And we now have unpublished data on the best inhibitor yet of that enzyme. If any of these molecules were to advance, if I'm allowed to dream, and help people, that would just be fantastic.

ZIERLER: In what form? Are we talking about medicines? In therapies? All of the above? What would it look like?

GREENBERG: At a minimum, they would be molecules that were useful in cells for people who are carrying out other studies to use as tools. That would be the minimum. The great dream would be to discover a therapeutic.

ZIERLER: This has been a wonderful conversation. I want to thank you so much for spending this time with me. I really appreciate it.

GREENBERG: Thank you.

[END]