Inhibitors of SAMHD1 Obtained from Chemical Tethering to the Guanine Antiviral Acyclovir

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is an enzyme with diverse activities. Its dNTPase activity degrades all canonical dNTPs and many anticancer nucleoside drugs, while its single-stranded nucleic acid binding activity promotes DNA repair and RNA homeostasis in cells. These functions require guanine nucleotide binding to a specific allosteric site (A1) on the enzyme. We previously described how the activities of SAMHD1 could be inhibited in vitro with fragment-based inhibitor design, using dGMP as a targeting fragment for the A1 site. However, these dGMP-tethered inhibitors had poor cell permeability due to the charged guanine monophosphate group. Here, we describe a new approach where the amino form of the guanine acyclic nucleoside acyclovir (NH₂-ACV) is used as the targeting fragment, allowing facile coupling to activated carboxylic acids (R-COOH), either directly or using linkers. This approach generates a neutral amide instead of charged monophosphate attachment points. High-throughput screening of a ∼375 compound carboxylic acid library identified two compounds (<b>8</b>, <b>11</b>) with similar micromolar affinities for SAMHD1. Compound <b>11</b> was obtained by direct coupling to NH₂-ACV, while compound <b>8</b> used a five-carbon linker. Both inhibitors had the same dibromonaphthol component from the carboxylic acid library screen. A crystal structure of a complex between SAMHD1 and <b>8,</b> combined with computational models of bound <b>11,</b> suggest how the dibromonaphthol promotes binding. The findings establish that guanine-based inhibitors targeting the A1 site do not require nucleotide or cyclic nucleoside structural elements. This guanine site targeting strategy is highly amenable to further chemical optimization.