The klaklak has revealed itself to be fatal to the mitochondria. And, when someone upsets the cell’s turbine, the result is normally catastrophic: it unchains a succession of programmed events known as apoptosis, which results in the cell’s death. “So then the idea came to us of tele-guiding the klaklak using a Zip Code that was not just specific but could also transport it to the inside of the target cell”, explained Dr. Renata during an interview by e-mail. “Thus we gained two levels of specificity. The first is the Zip Code and the second is the fact that the klaklak would not do anything if it did not arrive at a mitochondria, and for this reason it had to be positioned inside”. If you were to think on GRP-78 as a Zip Code (CEP in Portuguese): Dr. Renata is accustomed to responding to questions about her work in English, her research language – it’s the king and hits the nail on the head.
At this point, however, the postal analogy becomes less fruitful, because of the difficulty of the visualization of an important element in the strategy, which is the interaction between the two molecules. Whilst the numbers of a Zip Code and the letters of a postal address are symbols associated by convention, with a street or a building, the address that is printed on the anti-tumor letter bomb needs to literally enter into the addressee, or that is to say, into the part of the GRP-78 protein that sets itself up on the outside of the cell. The classical metaphor of this type of molecular recognition is that of a key in the lock, but a lock that has the peculiarity of sucking in the key and of whoever was behind on the other side of the door into which the key was fitted, only when the correct key has been inserted into it. Stated in another manner, when the complementary molecule of GRP-78 linked to the klaklak locks onto the receptor exposed by the stressed cell (or tumor), the GRP-78 sets off the molecular gearing that allows for the internal positioning of the klaklak, which for its part attacks the membranes of the mitochondria and in this manner starts the apoptosis.
The duo of Drs. Renata and Wadih have a major reputation when dealing with cell keys. In 2002 they had published in the magazine Nature Medicine an article dealing with repercussions, the fruit of a partnership with the Max Planck Institute of Molecular Genetics in Berlin. This was a type of micro-catalogue with molecular Zip Codes for five tissues: bone marrow, fat (adipose tissue), skeletal muscle, prostrate and skin. The catalogue was compiled with the help of a technique called phage display (something like an exhibition of phages), in which virus bacteriophages – bacteria parasites widely used in molecular biology experiments – are induced to exhibit on their surface parts of proteins chosen by the researcher. The duo of Drs. Renata and Wadih worked with tri-peptides, which are groups of three amino-acids, the structural units of proteins. They created a library – or virus library – with 47,160 types of phages, each one of them showing on the carapace a different tri-peptide, which in the experiment would be made of door knockers, billions of them.
The territory whose Zip Codes would be polled by the door knockers was the blood veins of a 48 year old man with terminal cancer and in a coma, after having suffered a cerebral hemorrhage, whose family agreed to collaborate with the test before the equipment that was keeping him alive was switched off. The legion of polling phages was injected into the patient, and fifteen minutes after his death, an analysis of samples of his tissue began, in order to determine the distribution of the tri-peptides (those that had locked themselves into the keyholes exposed by the cells of the blood veins appeared in greater number in the organ irrigated by the veins in question). In this manner the five groupings for the Zip Codes were discovered.
The proof that in principal these molecular Zip Codes of tissues could be used in tele-guided therapies was obtained in 2004, in a study carried out with mice and published in Nature. Using the codes of the molecular addresses for adipose tissue, the Brazilian researchers set off the klaklak upon it, killing the cells of the blood veins that had irrigated it. Without this source of supply, the cells of the fat began to die and were reabsorbed. The photos of the mice before and after the miraculous treatment against obesity captured the headlines in publications in the USA and Brazil. As the original idea was to apply the weapon against tumors, the attention fell again upon the Zip Code of the GRP-78, which another study group – this one was published during 2003 in Nature Biotechnology – indicated that it was not only highly specific for prostrate tumors but even more highly correlated with advanced cases and those of poor prognosis. The target for the letter bomb had been elected.
The results of the attack are those that came out in Cancer Cell. Drs. Wadih and Renata did not only reconfirm the GRP-78 as a valid Zip Code for localizing tumors but also of making use of three types of sample victims: lineages of tumor cells of the prostrate and breast; samples of tumors removed from patients and animal models of human tumors (mice transplanted with human tumor cells). “It’s been very efficient up until now and we believe that to target this protein could also work in other types of tumors”, stated Dr. Renata in a communication from the M. D. Anderson Cancer Center given out at that time. Since then she has confirmed that similar results have been obtained with lung cancer samples.
On the path of purity
There is a long trail forward before reaching the phase of clinical tests with human beings, by way of FDA authorization, the food and drugs agency in the United States. Firstly the group has to obtain extremely pure samples of the letter bomb, or that is to say, of the compounds that couple together to make the peptide groups capable of recognizing the GRP-78 along with the inducer of klaklak apoptosis. With these substances at the degree of purity demanded, they can then begin the other pre-clinical tests required by the FDA. “If everything goes to plan and we have a little bit of luck, 2006 will be the year in which we’ll arrive at the point of a phase 1 trial”, affirmed researcher Pasqualini. “Most certainly the M. D. Anderson Cancer Center, the doctors and the scientists involved are doing everything possible for this to occur as soon as possible.”
An obstacle on the path of this strategy to attain the condition of intelligent therapy against cancer is the question of toxicity, or that is, the possibility that the klaklak also breaks down the mitochondria of cells that are outside of the tumor. In spite of the high specificity of the GRP-78 and of the need for it to be projected onto the cell’s surface (which only occurs under stress), nothing guarantees that the letter bomb will not reach the cells of innocent and vital tissue. In the experiments, they annihilated cultured cells of normal tissue. Dr. Renata emphasized that, in this type of in vitro culture, the cells are found in a state of chronic stress, whilst in the body there is very little GRP-78 exposed to the peptide knockers in circulation. “We don1t forecast significant toxicity with this action. Nevertheless, we won’t know until we amplify our pre-clinical studies beyond that which was carried out with mice and with therapeutic doses, which don’t appear to be toxic.”
Drs. Renata and Wadih are maintaining active collaboration with researchers in Brazil. Of the seven authors of the article in Cancer Cell, four of them are Brazilians: as well as the couple, Marco Arap, Dr. Wadih’s cousin who was incorporated into the M. D. Anderson Cancer Center team contributed, and also Alvaro Sarkis, from USP’s Faculty of Medicine (FMU/USP), who provided “valuable human samples for the validation of the expression of GRP-78”, in the words of Dr. Renata. This noted Brazilian researcher, now settled in Texas, also stated that she is enthusiastic with the assistance that she has been receiving from Zip Code CEP 05403-010, of the FM/USP: Emmanuel Dias-Neto, from the Neuroscience Laboratory of the Psychiatry Institute, accompanied by Dr. Diana Nunes, also a specialist in genomics. Starting from January, the inventor of the Orestes methodology – the main technical innovation that came about during FAPESP’s genome projects – can be found at USA Zip Code 77030 in Houston, Texas.
The difficulty confronted by researchers such as Renata Pasqualini and Wadih Arap, who have been investigating new medicines against cancer, may well be compared with someone who needs to send a letter but does not know the name of the street or even the Zip Code of the person who is to receive. In order to be certain that a drug will reach tumor cells, they could remit thousands of letters to all of the town’s inhabitants – the patient, by analogy -, in the hope that some of them would reach the hands of the right people, or that is to say, the cancerous tissue. What in the world of the post office would represent paper wastage or spam in electronic mail, in the physiology of the sick person it is toxicity, the damage caused by the medicine in cells and tissues that have nothing to do with the illness. However, in their work at the University of Texas M.D. Anderson Cancer Center Renata and Wadih believe that they have discovered the Zip Code for some guys who watch over the prostrate, breasts and lungs, and are now preparing to combat them with, would you believe, letter bombs.
The analogy with the Zip Code is not new and has been in use since at least 2002 in order to describe the pioneering work of this Brazilian couple who only met when in the United States, even though both of them had studied in Sao Paulo under the oncologist Ricardo Brentani. Also, as a strategy the idea of tumor targeted therapy has been in use at various laboratories throughout the world, with different weapons, ammunition and bullet size. The innovation of researchers Wadih and Renata lies in the letter bomb that they put together and gained them notoriety in the magazine Cancer Cell in September of 2004, an ingenious coupling of two molecules capable of recognizing and preferentially killing tumor cells. If something produced in Texas truly merit the name of a “smart weapon”, then this technology must be among them, should the results obtained up until now in the culture cells and in animal models be replicated with human patients. The team is hopeful, if everything continues to go well, of beginning a clinical phase 1 test at the end of 2006, the type of study in which only a few patients will participate, simply to define if the new drug can be used with safety on human beings.
At the heart of the principal component of the device lies the protein No. 78 regulated by glucose, or GRP-78 (in abbreviated form). This molecule is produced in large quantities in cells that are found to be under stress, like those which for any reason find themselves deprived of oxygen (hypoxia) or of glucose. This is the case of tumor cells, which proliferate in hypoxic environments – hence the importance of vascularization in order to keep their growth from getting out of control – and produced lots of GRP-78. According to Renata, this specific relationship between the level of this protein and tumors was first demonstrated by Amy Lee, from the University of California in the city of Davis. Lee marked a DNA sequence that acts as an expression promoter (reading) of the gene corresponding to protein GRP-78 with another gene that, when read in conjunction, produces a blue protein (lacZ), thus dying and denouncing tissue with high expression levels of GRP-78. The experiment verified that the tumors became blue, with low expression levels of normal tissue.
Besides the high specificity for denouncing cancerous cells, the GRP-78 has a characteristic that has revealed itself to be of major interest for the development of a teleguided therapy: produced in the cells under stress, it does not remain confined to its interior, but in fact is anchored to the membrane. In other words, the protein is visible for the recognition of other molecules in the extra-cellular medium. “This was an important discovery, because it means that the protein could be accessible to a medicine projected to lock onto it”, says Arap in a bulletin written at the M.D. Anderson Cancer Center. “It’s a lot easier to target a protein on the outer cell than to send medicines into”. It is not known as yet what is the exact function of GRP-78 in the cell under stress, but one of the hypothesis is that it plays the role of alerting the organism’s immune system about the need for help, probably as an whole member of a machine charged with leading antigens (particles capable of deflagrating the production of antibodies) to the surface of the cell.
The other component of the device that annihilates tumor cells is a molecule in the form of a corkscrew dubbed klaklak, which plays the role of the explosive charge. It was discovered some years ago as an antibiotic, thanks to its capacity of demolishing bacteria membranes. When Renata and Wadih first met up in California at the Burnham Institute (their move to Texas took place in 1999), other researchers had suggested to them that the venom was also effective on human tissue, attacking mitochondria, the cellular organelles involved in the generation of energy and for this reason frequently described as the cell’s powerhouse. In Renata’s explanation, this hypothesis came about because the membranes of bacteria and of mitochondria have some similarity – a possible leftover of the origin of these organelles. According to the theory of endosymbiosis (proposed at the beginning of the 20th century and considered a type of delirium even after its revision and popularization by Lynn Margulis in the decade of the 1980’s), mitochondria are ancestral bacteria that at some point in evolutionary time had been incorporated by more complex cells.