Abstract Mastocytosis is a term used to describe a set of rare diseases that are characterized by increased mast cell development and the enhanced presence of mast cells in various tissues and organs. Once activated, mast cells promote inflammation through their robust production of histamines, leukotrienes, prostaglandins and many other effector molecules that promote itching, burning, smooth muscle contraction and life-threatening anaphylaxis, all of which are common symptoms of mastocytosis. Although patients suffering from mastocytosis are prescribed antihistamines and mast cell stabilizers, treatment options are limited, and more severe forms of the disease are extremely difficult to treat and remain life-threatening. Further, an incomplete understanding of the factors that regulate mast cell development has dramatically limited the ability to design novel therapeutics that selectively target mast cells. NemaGen’s published and preliminary studies have revealed that the enzyme carbonic anhydrase (Car)1 is exclusively expressed by mast cell progenitors. Further, we have demonstrated that small molecule-mediated inhibition of Car1 is sufficient to prevent murine and human mast cell development. In addition, our data demonstrate that in vivo treatment with known carbonic anhydrase inhibitors is sufficient to prevent mast cell development and inflammation in a murine model of mastocytosis. Collectively, these studies suggest that Car1 represents a novel therapeutic target for the treatment of mastocytosis and mast cell-mediated inflammation. Although existing carbonic anhydrase inhibitors are available, our studies have shown that their effective doses far exceed those required for translational applications. To address this, we have generated a strong team and effective workflow that allows us to synthesize and test new Car enzyme inhibitors. Further, our data demonstrate that we can generate significantly more effective inhibitors than currently available options. These exciting findings form the foundation of this SBIR proposal and two specific aims: (i) Design, synthesize and optimize novel carbonic anhydrase inhibitors. (ii) Evaluate the effects of Car enzyme inhibitors on enzymatic function and mast cell development both in vitro and in vivo. Collectively, these pre-clinical studies will allow us to generate new Car enzyme inhibitors for the treatment of mastocytosis and other mast cell-related disorders.