PROJECT SUMMARY Cellular proliferation increases demand for ribonucleotide pools to provide precursors for increased RNA, DNA, and membrane synthesis. This demand exceeds the capacity of uptake and salvage pathways, and must be met by increased de novo biosynthesis. In particular, lymphocyte proliferation is dependent on increased nucleotide levels, and targeting nucleotide biosynthesis pathways is the basis for a number of highly successful immunosuppressive treatments. CTP Synthase (CTPS) is the key regulatory enzyme in pyrimidine nucleotide biosynthesis. Humans have two CTPS isoforms encoded on separate genes, CTPS1 and CTPS2. While CTPS1 expression is generally increased in proliferative tissues, little else is known about differential roles of the two isoforms or how they are regulated. CTPS1 plays a critical role in the immune response, and loss of function mutations in humans cause severe immunodeficiency. Loss of CTPS1 results in impaired T and B-cell proliferation, but does not have deleterious effects in other human tissues, indicating unique dependence of the immune response on CTPS1 function. Selective inhibition of CTPS1, therefore, is considered a potentially powerful approach to immunosuppressive therapies with limited off-target effects. A number of inhibitors that specifically target CTPS1 have recently been reported, but the mechanisms of inhibition remain unclear. Here, we focus on the structural and functional characterization of CTPS1 and CTPS2 regulation by native allosteric regulators and by selective small molecule inhibitors. This work will provide insight into the control of nucleotide biosynthesis during cellular proliferation, and serve as the basis for design and targeted discovery of novel compounds with potential for immunosuppressive therapies.