Highlights d Developed high-throughput assays for SpCas9 and performed a small-molecule screen d Identified reversible and cell-permeable inhibitors that disrupt SpCas9-DNA binding d Inhibitors allow dose and temporal control of (non)-nucleasebased SpCas9 systems d Identified a pharmacophore for SpCas9 inhibition using structure-activity studies

The AID/APOBEC family enzymes convert cytosines in single-stranded DNA to uracil causing base substitutions and strand breaks. They are induced by cytokines produced during the body’s inflammatory response to infections, and help combat infections through diverse mechanisms. AID is essential for the maturation of antibodies and causes mutations and deletions in antibody genes through somatic hypermutation (SHM) and class-switch recombination (CSR) processes. One member of the APOBEC family, APOBEC1, edits mRNA for a protein involved in lipid transport. Members of the APOBEC3 subfamily in humans (APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D/E, APOBEC3F, APOBEC3G and APOBEC3H) inhibit infections of viruses such as HIV, HBV and HCV, and retrotransposition of endogenous retroelements through mutagenic and non-mutagenic mechanisms. There is emerging consensus that these enzymes can cause mutations in the cellular genome at replication forks or within transcription bubbles depending on the physiological state of the cell and the phase of the cell cycle during which they are expressed. We describe here the state of knowledge about the structures of these enzymes, regulation of their expression, and both the advantageous and deleterious consequences of this expression including carcinogenesis. We highlight similarities among them and present a holistic view of their regulation and function.

Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small molecules (PHICS), which enable two example kinasesAMPK and PKCto phosphorylate target proteins that are not otherwise substrates for these kinases. PHICS are formed by linking small-molecule binders of the kinase and the target protein, and exhibit several features of a bifunctional molecule, including the hook-effect, turnover, isoform specificity, dose and temporal control of phosphorylation, and activity dependent on proximity (i.e., linker length). Using PHICS, we were able to induce native and neo-phosphorylations of BRD4 by AMPK or PKC. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target protein Bruton’s tyrosine kinase in cells. We envision that PHICS-mediated native or neo-phosphorylations will find utility in basic research and medicine.

Human APOBEC3B deaminates cytosines in DNA and belongs to the AID/APOBEC family of enzymes. These proteins are involved in innate and adaptive immunity, and may cause mutations in a variety of cancers. To characterize its ability to convert cytosines to uracils, we tested several derivatives of APOBEC3B gene for their ability to cause mutations in Escherichia coli. Through this analysis, a methionine residue at the junction of amino- and carboxy-terminal domains was found to be essential for high mutagenicity. Properties of mutants with substitutions at this position, examination of existing molecular structures of APOBEC3 family members and molecular modeling suggest that this residue is essential for the structural stability of this family of proteins. The APOBEC3B carboxy-terminal domain (CTD) with the highest mutational activity was purified to homogeneity and its kinetic parameters were determined. Size exclusion chromatography of the CTD monomer showed that it is in equilibrium with its dimeric form and MALDI-TOF analysis of the protein suggested that the dimer may be quite stable. The partially purified amino-terminal domain did not show intrinsic deamination activity and did not enhance the activity of the CTD in biochemical assays. Finally, APOBEC3B was at least 10-fold less efficient at mutating 5-methylcytosine (5mC) to thymine than APOBEC3A in a genetic assay, and was at least 10-fold less efficient at deaminating 5mC compared to C in biochemical assays. These results shed light on the structural organization of APOBEC3B catalytic domain, its substrate specificity and its possible role in causing genome-wide mutations.