**4. Historical background for drug design**

Now, you know why Sir Winston Churchill, who smoked cigar all his life, never developed cancer, but film actor Yul Brenner smoked cigarettes and died of lung cancer. If you were to analyze their urine samples, you will find that Mr. Brenner urine contains nicotine N-oxide. The gene monoamine oxidase is activated in Brenner to make nicotine N-oxide not in Sir

**Figure 1.** NIH Scientific Achievement Award. Aziridines as single strand DNA binding agent.

While I was busy designing drugs, such as AZQ, to shut off genes which cause brain cancers, my colleagues in the other labs at NCI (National Cancer Institute) have isolated hundreds of chemicals from the tobacco tar which contains dozens of carcinogenic chemicals. If you would apply the tobacco tar on the skin of mice, within a few weeks, tumor develops on the skin surface. The major culprit is nicotine which is considered as one of the most addictive chemicals. Some studies showed that it is even more addictive than many known narcotics such as marijuana, opiates, and heroin. Oral cancer (OC) is caused by chemicals released by chewing tobacco. Most football players chew tobacco; they call it smokeless tobacco. Smoking burns tobacco generating even more aromatic amines which are known carcinogens. Nitrosoamines

After the completion of the Human Genome Project, we have identified specific mutations responsible for a specific disease. Now, we design drugs to attack that specific mutation to shut off that gene. The completion of the Human Genome Project has the greatest impact on

Winston. While Sir Winston lived, Brenner died.

42 Prevention, Detection and Management of Oral Cancer

bind to DNA producing mutations.

On the other hand, if cancer is caused by multiple mutations, we could use drug therapy by preventing malignant cell replication developed by Ross by cross-linking both strands of DNA. Using dyes specific to OC cells as carriers for nitrogen mustard, as done by Ross in making Melphalan, we could also develop new class of drugs to attack cancer cells in the other parts of the oral cavity. The bad news is that there have been 13 different forms of oral cancer identified. The good news is that for designing a drug, we have to find a dye which colors one of these tumors. There are hundreds of dyes available for testing. Once we succeed in finding a dye, we could design drugs by using our method by attaching aziridines to attack that specific oral cancer by shutting off mutated genes by binding to a single strand of DNA. What would happen if we succeed and when next-generation sequencers produce inexpensive and fast sequencing genomes is that we could identify all mutated genes on all 13 oral cancers with precision and accuracy and design drugs to shut off those genes.

In the laboratory of the Sir Walter Ross at the Royal Cancer Hospital of London University, England, I was trained to design drugs to attack mutated DNA shutting off mutated genes. Professor Ross had spent all his life working on "Biological Alkylating Agents" and published a series of paper including a book [9–13]. Using the same rationale, I worked with Professor Ross for almost 10 years at London University developing anticancer drugs. Instead of crosslinking DNA with nitrogen mustards, I used aziridines to bind to a single strand of DNA shutting off the genes.

During 10-year period in the Professor Ross' Lab, I made over 120 such drugs to attack a solid tumor called the Walker carcinoma 256 in rats. The most effective drug was called CB 1954 (2-4, dinitrophenyl aziridinyl benzamide) (see structure in **Figure 1**). It was 70 times more toxic to the tumor cell [14]. It is the most effective drug ever made against the solid experimental tumor for which I was honored with the Royal Cancer Hospital's Institute Cancer Research postdoctoral award. Why mutated DNA must be attacked is because mutated DNAs code for wrong amino acids which produce wrong protein and cause abnormal growth leading to cancers. The reason why we work on mouse model is that if you would compare the genome of man, mouse, and monkey, they are all mammalians and their genomes are very similar. Once you succeed in attacking mouse tumor, it opens gate to attack human tumors. Now, you know how challenging it is to shutting off a mutated gene and how easy it is to introduce mutations in the same gene by smoking.

Walker carcinoma 256 in rats or glioblastoma in humans. It would be much easier to attack carcinoma. And also suppose that I find a single dye which specifically colors a specific carcinoma cell. Now, my work begins to attach aziridines or carbamate or both as we succeeded in making AZQ for attacking brain tumor. Nowadays, I have to submit the research proposal to the Safety Committee (IRB: Institutional Review Board) for their approval. Since I will be

The Impact of Sequencing Human Genome on Drug Design to Treat Oral Cancer

http://dx.doi.org/10.5772/intechopen.80231

45

Firtz Heber, a German Army officer, worked on the development of chemicals as a weapon of war. He was responsible for making deadly nerve gases and nitrogen mustards. Before the WWI, he was honored with a Nobel Prize for capturing nitrogen directly from the atmosphere by burning the element magnesium in the air forming its nitride. Upon hydrolysis, nitride is converted to its nitrate which is used as a fertilizer. Using this method, we could make unlimited amount fertilizer. Nitrate is also used for making explosive. Soon after the WWI, Heber was charged with a crime against humanity for releasing hundreds of cylinders of chlorine gas on the Western front killing thousands of soldiers in the trenches. When allied forces reached his residence, his son shot himself and his wife committed suicide. Heber went in hiding in Switzerland. After the war, German government got his release as a part of the peace negotiations. Heber returned home to hero's welcome. Although he promised never to work on the nerve gases again, secretly he continued to develop more lethal analogs of highly toxic nitrogen mustards. It was Heber who first made the notorious bis-dichloroethyl methyl amine. Because it smells like mustard seeds, it is called as nitrogen mustard. During the next 20 years, before the beginning of the WWII, hundreds of more toxic analogs of nitrogen mustard were developed. The bad news is that they are highly toxic and the good news is that

Nitrogen mustard was mercilessly used during the WWII by both German and Italian armies against allied forces. Most soldiers exposed to nitrogen mustard were frozen to death. Their blood analysis showed a sharp decline in white blood cell (WBC). Since patients with the cancer of the blood called leukemia showed a sharp increase of WBC, Professor Ross and his group wondered if minimum amount of nitrogen mustard could be used to control leukemia in cancer patients. It was a success. For the following 30 years, Ross developed hundreds of derivatives of nitrogen mustard to treat a variety of cancers. His most successful drugs are

Radiolabeled study showed that nitrogen mustard shut off genes by binding to DNA by crosslinking. At London University, I work for Professor Ross for almost 10 years first as his graduate student, then his post-doctoral fellow, and then as his special assistant. I worked with the deadliest nerve agents such as nitrogen mustards, carbamates, and aziridines developed during Hitler's time for evil purposes. We are converting evil into good. These agents easily

using highly toxic nerve gases and nitrogen mustard, the proposal will be rejected.

**5. Drug design for treating cancer**

they shut off genes.

**6. Rationale for developing anticancer drugs**

chlorambucil, melphalan, and merophan [2–6, 14].

The good news about smokers is that they could see their own genome on their computers and could also see the progress of mutations of their own genomes before and after smoking. Now, I do not have to tell my best friends not to smoke. All I have to do is to give the two CDs of their genomes and let them see for themselves. Let them see on their own computers and compare the two genomes. First, CD taken soon after their birth and second taken after they become smokers. Before I talk about the sequence-specific tumors of oral and lung cancers, let me share with you how our genome, the book of our life, looks like before you smoke.

The basis of OC is that people who are chewing tobacco or inhaling burning tobacco by smoking (as in India) or chewing betel quid, betel nut, etc. (as in Taiwan) causing major mutations in their genomes producing a host of chemicals which damage the normal function of the cell causing them to become abnormal or cancerous. To understand the molecular basis of cancers, we have to sequence the normal as well as cancer cell genomes for comparison.

To refresh your memory, I repeat. Carcinoma is the most common type of cancer. It begins in the epithelial tissue of the skin, or in the tissue that lines the internal organs, such as mouth, oral cavity, reparatory tract, and lungs. There are 220 different types of tissues in the normal human being. We have sequenced (read letter by letter the entire script of nucleotides, their numbers, and their orders in which they are arranged). After genome sequencing, we can compare with the sequence of oropharynx carcinoma with normal cell from nonsmokers. I am happy to inform you that we just completed the Thousand Genome Project. Now, we can compare thousands of the same mutated sequence a time. We can locate the specific mutations with precision and accuracy. To locate specific mutations, in all oral cavity cancers, similar comparison can be studied in several types of salivary gland cancers including adenoid cystic carcinoma, mucoepidermoid carcinoma, and polymorphous low-grade adenocarcinoma. Tonsils and base of the tongue tissues also develop lymphomas.

Once a mutation is identified in a specific oral cancer, my job begins trying to find a dye which colors these tumors. Once a dye is found, I could attach the toxic groups to shut off their genes. There are hundreds of dyes and there are hundreds of their combinations. With AT and GC, four nucleotides of DNA, I get 64 combinations; imagine how many combinations I get from hundreds of dyes. Fortunately, there are finite combinations. I could find it. Suppose I want to design drugs to treat cancers of the oral cavity. The cancer begins in the epithelial tissue of the skin, or in the tissue that lines the internal organs, such as mouth, oral cavity, reparatory tract, and finally reaching lungs. We have been designing drugs to attack toughest solid tumor like Walker carcinoma 256 in rats or glioblastoma in humans. It would be much easier to attack carcinoma. And also suppose that I find a single dye which specifically colors a specific carcinoma cell. Now, my work begins to attach aziridines or carbamate or both as we succeeded in making AZQ for attacking brain tumor. Nowadays, I have to submit the research proposal to the Safety Committee (IRB: Institutional Review Board) for their approval. Since I will be using highly toxic nerve gases and nitrogen mustard, the proposal will be rejected.
