The Maverick of Bethesda

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When the Merlino Laboratory became part of the newly formed Laboratory of Cancer Biology and Genetics (LCBG) in 2007, I heard about Dr. Peter Blumberg for the first time; not about his research but about his famous invention: squirrel-proof birdseed. It was made by treating birdseed with a compound from chili pepper, which burns the tongues of squirrels but not of birds. Another thing intriguing me was that his lab was full of Hungarian scientists. From my very good friend from Budapest, I learned that Hungarians love the red chili pepper - paprika - very much. They even have an institute dedicated to the study of chili peppers. Putting these two facts together, I imagined that Peter's research must be all about chili pepper, so he must have collaborated with Hungarian scientists, thus recruiting many Hungarians to his laboratory. It turned out that I got it all wrong. What led him to the study of the chili pepper compound was a more fascinating story.

To understand Peter's journey to that compound, we have to go all the way back to the 18th century. In 1775, Dr. Percival Pott in England found that "soot wart", a squamous cell carcinoma, happened frequently in the scrotal skin of young chimney sweepers. He hypothesized that the cancer was associated with occupational exposure to soot. However, no one could prove it for the following 140 years until 1915, when Yamagiwa and Ichikawa showed that daily coal tar treatment induced tumor formation in rabbit ears. This was the very first tumor induction animal model in the history of cancer research. Their experiment opened the new research field of chemical carcinogenesis.

Subsequently, many efforts were made for isolating carcinogenic chemicals from coal tar. That led to the discovery of the famous 7,12-dimethylbenz[a]anthracene (DMBA). Over the next decades, researchers tried to figure out the effect of these chemicals on cells. In the 1940s, Mottram and Berenblum used mouse studies to show that some chemicals could induce tumors by themselves ("tumor initiator"), while others could only enhance the carcinogenic effect ("tumor promoter"). This model eventually evolved to the multi-stage carcinogenesis hypothesis, which still guides cancer research today.

Mottram and Berenblum used DMBA as the tumor initiator and croton oil as the tumor promoter. In the 1960s, Hecker isolated 12-0-tetradecanoyl phorbol-13-acetate (TPA) from croton oil and proved its tumor promoter activity. TPA became one of the most well-known compounds in cancer research, and the series of its analogous compounds was generally referred to as phorbol esters.

In 1965, Brookes and Lawley found that tumor initiators covalently bound to DNA in mouse skin and were DNA-mutating agents. The effect of tumor promoters on the cells, however, was a topic of hot debate. Some said they induced cell proliferation, and others believed that inflammation was the key. No matter what, phorbol esters have become the standard tumor promoters used in carcinogenic studies.

As soon as he started his own lab, Peter aimed to solve the mystery of the tumor promoters. Being a biochemical pharmacologist, the question for him was what the cellular receptors of TPA, or phorbol esters, were. He set a straightforward strategy: treating the cells with radiolabeled phorbol ester that he had synthesized, and then looking for specific binding. However, there were many technical challenges. A critical problem was that TPA was extremely lipophilic. Therefore, most of the TPA added to cultured cells would be absorbed non-specifically into the cell membrane, obscuring the small amount that would bind to its receptor. To resolve this problem, Peter thought of finding another phorbol ester analogue exhibiting lower lipophilicity AND stronger potency.

The first part is easy; you can predict the solubility of a compound from its structure. The second part is tricky: how to measure the potency of compounds? Peter adopted an assay well known among natural product chemists for measuring the activity of compounds, the smallest dose to induce mouse ear reddening (inflammation). The literature reported that the phorbol related compound resiniferatoxin (RTX), derived from Euphorbia resinifera (a cactus-like plant commonly in Morocco), was 1000-fold stronger than TPA in the mouse ear inflammation assay!

However, when Peter tested RTX in cultured cells, the results were not what was expected. RTX could not induce two standard responses to TPA, increasing deoxyglucose transport and decreasing cell surface glycoprotein levels. Obviously, RTX was not what he was looking for.

Peter moved on to assess other phorbol esters and identified phorbol 12,13-dibutyrate (PDB), which met the need of a phorbol derivative with high potency but much lower lipophilicity. Using the radio-labeled PDB, he was able to demonstrate the existence of the TPA receptor [1]. As he characterized the receptor, marked similarities emerged between the phorbol ester receptor and protein kinase C (PKC) [2], which was being characterized by Nishizuka’s group at Kobe University, Japan, at that time . These parallels led Nishizuka to show that the TPA receptor was PKC, which was the first characterized molecular target for tumor promotion in skin [3].

The following years were the golden time of PKC research. Nishizuka’s group demonstrated that diacylglycerols, the breakdown fragment of membrane lipid, could activate PKC. Peter further proved that it was the actual endogenous ligand of PKC. From the structures, TPA was obviously an analog of diacylglycerols. For the first time, cancer researchers could investigate the signaling pathways of tumor promotion. Moreover, many PKC isoforms were identified, and their tissue distribution was characterized. A new field was opened in cancer research, leading to many therapeutic opportunities. Peter kept working on the phorbol ester derivatives and PKC signaling in the next few years. He was well recognized as one of the top PKC experts worldwide.

However, Peter never forgot RTX. He still wondered what it was actually doing, if it had nothing to do with PKC. In his own words, he had the answer, now he needed to know what the question was.

At this time, Arpad Szallasi came to Peter's lab from Hungary. His first assignment from Peter was to figure out the target or receptor of RTX. A starting point was its molecular structure. RTX differed from typical, potent phorbol esters by having a substituted phenylacetate ester at a position where a free hydroxyl group was an essential element of the pharmacophore for the typical phorbol esters. However, the moiety of the substituted phenylacetate ester was identical to a critical part of the pharmacophore of capsaicin, the compound responsible for the tongue-burning feeling of chili pepper. In fact, Peter had been aware of the structural homology between RTX and capsaicin for years, but it had taken him a decade to obtain a sufficient supply of the compound to study it in detail.

Arpad and Peter soon found that treatment of RTX caused hypothermia, neurogenic inflammation, and pain in rat, very similar to capsaicin but much stronger. These effects implied that RTX, like capsaicin, acted on neurons. In 1989, nine years after finding that RTX was not the solution for demonstrating the phorbol ester receptor, the true target for RTX was identified; it was an ultrapotent capsaicin analog [4].

This opened a new era of Peter's research, doing neurobiology at the National Cancer Institute. First he demonstrated that RTX had no tumor promoting effect, so its receptor was totally different from PKC. He and Szallasi then applied again the strategy of using radio-labeled compound to identifying a RTX receptor and characterizing its specific expression in tissues. This receptor can bind capsaicin analogues, so he named it the "vanilloid receptor", following the fact that these compounds possess a vanillyl group [5]. Moreover, Peter found that the RTX was such a strong stimulant that it could effectively desensitize neurons for very long time- in other words, you can use RTX as a painkiller!

Living on a farm, Peter was aware that squirrels always got on feeders to steal birdseed. They were too gymnastic to be blocked by a simple device, like cover caps. This question made Peter think about the difference between squirrels and birds, and he recalled his research data instantly: the vanilloid receptor was expressed on the neurons of mammals, not birds. How about treating birdseed with capsaicin compounds? Squirrels would find the tongue burning taste and avoid it, while birds would not feel the difference. Peter invented squirrel-free birdseed, and NIH profited by licensing it. This invention was reported widely in the media: a NIH scientist used chili pepper to protect bird food from looting by squirrels! Outside the campus, Peter was well known as the "spicy birdseed scientist" [6].

Not being limited to the roles as a biochemist, pharmacologist, and cancer researcher, Peter had expanded his interests into neuroscience researcher and even evolutionary biologist.

These results soon attracted the attention of neuroscientists. For a long time, they had been searching for the mechanism of pain sensing. Now they could not believe that it was there: from the effect of capsaicin and RTX, the vanilloid receptor should function for sensing pain. What an embarrassment! A major advance in neuroscience was being made by a cancer researcher, not by a neuroscientist.

Naturally, the next step was to identify the gene of this receptor, and quite a few people were chasing it. One of them was David Julius, who designed a very smart approach. From the studies of Peter and Szallasi, he realized that the expression of vanilloid receptors was enriched in dorsal root ganglion. His group prepared a cDNA library from the neural tissue, generating pools of 16,000 clones in total. The standard epithelial HEK293 cells were transfected with the DNA pools, treated with capsaicin, and watched for reporter activity under fluorescence microscope. Through this laborious process, the gene of the vanilloid receptor were identified [7]. It was later named transient receptor potential vanilloid subfamily member 1 (TRPV1).

The characterization of the TRPV1 gene opened the door toward a new research field - molecular mechanisms of "noxious" sensation (pain, heat, tissue damage, etc.). First, Julius showed that TRPV1 also detected heat. Second, he found homologues of TRPV1, forming a family of "nociceptors". Some of them sense cool temperature, others detect stimulant compounds such as menthol or mustard oil. All these discoveries have significant clinical implications in pain control and thermoreguation.

Instead of studying the molecular biology of the TRPV family, Peter was more interested in the pharmacological aspects of the vanilloid receptors, especially identifying their expressing sites, agonists and antagonists, and opportunities for clinical application. He promoted the idea using the neural desensitizing effect of RTX for pain control.

Since the discovery of TRPV1, the research on the molecular mechanism of pain and thermosensation has grown exponentially. It now has become a significant research area in neuroscience, and its potential in clinical application is well recognized. Those who got into this field at an early stage, like Julius and Ardem Patapoutian, have gotten great academic recognition. Not so many remember it actually was Peter who actually discovered the capsaicin receptor.

He did not really care. He liked to build a bandwagon instead of jumping on one, and now he moved to build the next one. Although the clinical potential of the vallinoid receptor was highly regarded, only a few highly potent agonists and antagonists were available for testing at the time. In collaboration with Jeewoo Lee at Seoul National University he continued to test more compounds and their pharmacological effects. As most people - including neuroscience researchers- have enthusiastically focused on antagonists of the vallinoid receptor for pain control, he has tried to think differently. Years ago he developed the idea that the strong neural desensitization effect of RTX could be used to block pain. Unfortunately, his patent for use of RTX for desensitization of pain (and other actions of the capsaicin receptor) fizzled in a failed development effort by the NIH licensee (with whom he was not allowed to advise because of a conflict of interest !). Given the impact of pain on American health, this has been a source of great regret for him. Yet others at the NIH continue to study resiniferatoxin for pain in clinical situations, neglecting to give Peter his due recognition. [8]

Since Szallasi's arrival many years ago, Peter has built a consistent connection with scientists from Hungary. When one was leaving, another would find his laboratory. Over the years, Peter got familiar with Hungarian language. He liked to say, to solve a problem you have to throw every kind of knowledge together into one big pot, just as in goulash. He pronounced Budapest as Budapesht, like the Hungarians do.

He also learned another language- sign language, and can communicate with it passibly. Believing that people should be evaluated on their job-relevant abilities, not on job-irrelevant traits, he recognized that the deaf community offered a pool of talented scientists who might be being ignored by others. Over the years, Peter has had 22 deaf or hard-of-hearing scientists in his group, publishing some 70 papers. This initiative has now been continued by Kent Hunter.

In 2018, Peter decided to retire. He had been a NIH investigator for more than three decades, but life is bigger than just science. LCBG organized a farewell party to celebrate his scientific achievements; now he has the time and freedom for his other interests. He is living it up on his farm, takes care of his horses, and keeps exploring the new frontiers in his life.

In 2021, news came out that the Nobel Prize in Physiology or Medicine was awarded to David Julius and Ardem Patapoutian "for their discoveries of thermal and mechanical transducers", with David Julius being credited with discovering the capsaicin receptor. Many of Peter's friends were very surprised, because they knew who discovered TRPV1, the very first one identified in the thermal/mechanical receptor family. Some of them were outraged, even saying that Peter was "cheated". Peter kind of shrugged it off. He said, to be fair, Julius cited his studies [7][9]. Peter thought that this Nobel Prize was intended to highlight an important advance in neuroscience research, and he is not a neuroscientist.

In fact, this is not the first time that a Nobel Prize excluded the "outsider", nor probably the last time. Yamagiwa Katsusaburo and Koichi Ichikawa built the very first animal model of cancer, identified the first carcinogenic substance, and laid the foundation for the field of chemical carcinogenesis. However, Nobel Committees skipped them and gave the award to Johannes Andreas Grib Fibiger for his alternative carcinogenic discovery: a microbial parasite as the cause of cancer. Unfortunately, this "finding" was discredited by other scientists shortly afterward. Many people believe that Yamagiwa and Ichikawa were not awarded a Nobel Prize because they did not belong to the circle of European scientists in cancer research.

That's OK. Peter likes to think out of the box, so he never belongs to any circle. He has been the Maverick of Bethesda and will keep it the way it is.

 

 

[1] https://pubmed.ncbi.nlm.nih.gov/6941848/

[2] https://pubmed.ncbi.nlm.nih.gov/3456269/

[3] https://pubmed.ncbi.nlm.nih.gov/20668066/

[4] https://pubmed.ncbi.nlm.nih.gov/2747924/

[5] https://pubmed.ncbi.nlm.nih.gov/2174484/

[6] https://www.washingtonpost.com/local/when-youre-hot-youre-hot-meet-the-man-who-invented-chili-pepper-birdseed/2016/04/12/b06dc402-fff7-11e5-b823-707c79ce3504_story.html

[7] https://pubmed.ncbi.nlm.nih.gov/9349813/

[8] https://irp.nih.gov/blog/post/2022/05/pain-research-center-accelerates-irp-pain-studies

[9] https://www.painresearchforum.org/forums/interview/149899-discoveries-trp-and-piezo-channels-pave-path-kavli-prize-conversation-david

 

 

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