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Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC 3 NH 2 ) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC 3 NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a ) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1- 5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.