Alan Clayton Sartorelli PhD
Alfred Gilman Professor of Pharmacology
Cancer research; Drug development; Drug resistance
Current ProjectsCurrent projects we are working on are:
- development and study of the molecular mechanism of action of agents inducing the terminal differentiation of neoplastic cells to adult forms with no proliferative potential;
- development and evaluation of inhibitors of the enzyme ribonucleotide reductase;
- development and mechanism of action of DNA base methylating and chloroethylating agents;
- studies on the mechanism of drug resistance;
- development of agents that preferentially attack hypoxic cells of solid tumors and an evaluation of the mechanism by which they express selectivity for oxygen-deficient cells;
- evaluation of the role of retinoic acid receptors in leukemia cell differentiation;
- studies of ABC transporters during embryonic development and multidrug resistance.
Ongoing research in Alan C. Sartorelli’s laboratory consists of several major areas concerned with the design, synthesis and preclinical evaluation of newly conceived potential antineoplastic agents, particularly concentrating on the molecular mechanism by which agents developed in his laboratory exert their anticancer effects; mechanisms by which malignant cells circumvent the cytodestructive actions of the newly developed antineoplastic agents; and means by which tumor resistance mechanisms can be circumvented.
Extensive Research Description
The emphasis of our research is being placed upon the following: (a) the most intense research ongoing in the laboratory involves the design of a new class of antineoplastic agents, the 1,2-bis(sulfonyl)hydrazines that function as alkylating agent prodrugs which target the O-6 position of guanine in DNA (a prodrug is an inactive form, which can be converted by neoplastic cells to an active therapeutic form). This research has led to the development of two prototype agents that following activation generate a second reactive species, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, abbreviated 90CE, with broad spectrum potent activity against transplanted murine and human tumors. The first series of synthetic compounds yielding a clinical candidate were 2-aminocarbonyl-1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines which undergo activation by base catalyzed decomposition to yield 90CE. The selected candidate agent, onrigin™ (formally called cloretazine), which chloroethylates the O-6 position of guanine in DNA and ultimately forms DNA G-C cross-links, has shown considerable activity in elderly patients with poor risk de novo acute myelogenous leukemia (AML) for which no standard therapy is available. Onrigin as a single agent has produced an objective response rate (CR, complete remission + CRp, all of the criteria for a CR with platelets less than 100,000/µl) of 34% these patients, with modest systemic toxicity.
A second group of 1,2-bis(sulfonyl)hydraznes that generates 90CE is exemplified by 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[(1-4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), which undergoes a different activation mechanism (bioreduction) to generate 90CE; this agent and several analogs thereof have been developed in our laboratory to target the reductive enzymatic potential of hypoxic regions within solid tumors. We have pioneered the concept, introduced in the early 1970’s, that hypoxic areas in tumors are sites of vulnerability in that the environment of these cells promotes selective reductive activation directly in solid tumors, creating an exploitable difference between normal cells in body tissues, which are usually well oxygenated, and hypoxic neoplastic cells. We have developed several lead prodrug compounds that selectively generate cytotoxic products that destroy hypoxic tumor cells, which are usually quite resistant to therapeutic intervention, as well as employing the hypoxic fraction to generate agents that circumvent the major resistance mechanism to agents targeting the O-6 position of guanine in DNA.
b. O6-Alkylguanine-DNA alkyltransferase (AGT), a repair protein that catalyzes the transfer of both alkyl and methyl groups from the O-6 position of DNA guanine generated from chloroethylating agents such as onrigin, KS119, and carmustine (BCNU), as well as temozolomide and other tumor methylating agents to cysteine 145 of the AGT molecule, thereby inactivating AGT and restoring the O-6 position of guanine to its native form. This action results in tumor reistance to the chloroethylating and methylating agents described above and, in experimental systems, a direct relationship exists between AGT activity and the degree of resistance to O-6 guanine targeting agents. AGT is also present in varying amounts in normal tissue, thereby protecting normal tissue to agents such as onrigin; however, the concentration of AGT in tumors often is significantly higher than in corresponding normal tissue.
One of the most potent known inhibitors of AGT is O6-benzylguanine (O6-BG); this agent reacts with AGT to form S-benzylcysteine in the active site of the protein. As a result, O6-BG depletes AGT and increases the sensitivity of tumor and host cells to agents that chloroethylate the O-6 position of DNA guanine and ultimately produce lethal DNA G-C cross-links. Non-toxic doses of systemic O6-BG have been shown in patients to deplete the AGT content of tumors and enhance the antineoplastic action of the nitrosourea carmustine. A similar depletion of AGT in normal tissue is produced by O6-BG; this action also sensitizes host tissue to the cytodestructive action of carmustine, necessitating an 80% decrease in the dose of carmustine because of excessive myelosuppression. The large decrease in the dosage of the nitrosourea results in an ineffective level of carmustine. To circumvent this problem, we are currently designing prodrug inhibitors of AGT activated by the reductive potential of hypoxic tumor cells in an effort to selectively deplete AGT in solid tumors relative to normal tissue, such that sensitivity to O-6 DNA guanine targeting agents will be selectively cytotoxic to solid tumors.
c. An additional series of agents that we have developed is exemplified by the potential drug, triapine, the most potent known inhibitor of the important enzyme ribonucleotide reductase (RNR); triapine has also shown clinical activity against acute myelogenous leukemia. RNR catalyzes a rate-limiting reaction in which ribonucleoside diphosphates are converted to their corresponding deoxyribonucleoside diphosphates, the precursors of the deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis and repair. Mammalian RNR consists of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. The R2 subunit of RNR maintains dNTP pools for DNA replication during the S phase of the cell cycle and p53R2 supplies dNTPs for DNA repair when DNA damage occurs. Since 50% of human cancers contain mutated or deleted p53 it was not clear how malignant cells that lacked inducible p53R2 supplied sufficient dNTPs for DNA repair. To study this issue, we employed HCT-116 p53(-/-) cells which we developed from p53(+/+) HCT-116 human colon carcinoma cells and demonstrated an accumulation of the R2 protein in these cells in response to triapine, cisplatin and doxorubicin. R2-targeted small interfering RNA (siRNA) was used to decrease the level of the R2 protein and caused a marked increase in sensitivity to the DNA-damaging agent cisplatin, as well as to the RNR inhibitor triapine. These findings and others provided evidence that R2-RNR was capable of supplying dNTPs for the repair of DNA damage in p53 deficient tumor cells. We are currently studying the role of the S phase checkpoint in facilitating DNA damage repair by R2-RNR in p53 deficient ovarian carcinoma cells damaged by cisplatin and other platinum-containing drugs, as well as by triapine, which preferentially depletes dATP levels. The mechanism involved in the preferential depletion of dATP is currently unknown.