IDENTIFICATION OF DRUG TARGETS THROUGH GENETICS

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The overall goal of our work is to use modern genetics to identify the target(s) of new drugs that show promise for the treatment of toxoplasmosis in AIDS patients. Our approach has been to develop a genetic map and appropriate methodologies that will enable us to identify and clone genes that in mutant form can lead to resistance to a given drug. For the future, we plan to continue and extend this genetic approach to map resistance genes for atovaquone, clindamycin and other promising, new drugs as they become available. To do this, we will first expand our set of restriction-fragment-length-polymorphism (RFLP) probes to include those representing novel organella genomes. Then we will map the resistance genes with respect to these and the nuclear RFLP markers. Transfection will be used to precisely localize the resistance gene. Sequence analysis will reveal its identity. Such as resistance genes are expected to include those coding for the primary target of the drugs in question and thus reveal their mode of action. This approach is applicable to any drug and has the power of being "assumptionless" (no guesses need to be made in advance about the likely target) and rapid. To date, we have: identified and characterized the physical linkage groups of T. gondii completed a low resolution genetic map for the nuclear genome. mapped genes responsible for resistance to two drugs (sinefungin and adenosine arabinoside) and begun the process for a third (atovaquone). developed protocols for transient and stable transfection of T. gondii. Identifying the target of a given drug is essential to knowing how to modify the drug to improve its efficacy, what other drugs might work and which drugs might act synergistically.