Structure-Guided Design of a Potent and Specific Inhibitor against the Genomic Mutator APOBEC3A

Abstract

Nucleic acid structure plays a critical role in governing the selectivity of DNA- and RNA-modifying enzymes. In the case of the APOBEC3 family of cytidine deaminases, these enzymes catalyze the conversion of cytosine (C) to uracil (U) in single-stranded DNA, primarily in the context of innate immunity. DNA deamination can also have pathological consequences, accelerating the evolution of viral genomes or, when the host genome is targeted by either APOBEC3A (A3A) or APOBEC3B (A3B), promoting tumor evolution leading to worse patient prognosis and chemotherapeutic resistance. For A3A, nucleic acid secondary structure has emerged as a critical determinant of substrate targeting, with a predilection for DNA that can form stem loop hairpins. Here, we report the development of a specific nanomolar-level, nucleic acid-based inhibitor of A3A. Our strategy relies on embedding the nucleobase 5-methylzebularine, a mechanism-based inhibitor, into a DNA dumbbell structure, which mimics the ideal substrate secondary structure for A3A. Structure–activity relationship studies using a panel of diverse inhibitors reveal a critical role for the stem and position of the inhibitor moiety in achieving potent inhibition. Moreover, we demonstrate that DNA dumbbell inhibitors, but not nonstructured inhibitors, show specificity against A3A relative to the closely related catalytic domain of A3B. Overall, our work demonstrates the feasibility of leveraging secondary structural preferences in inhibitor design, offering a blueprint for further development of modulators of DNA-modifying enzymes and potential therapeutics to circumvent APOBEC-driven viral and tumor evolution.

Seven-membered ring nucleobases as inhibitors of human cytidine deaminase and APOBEC3A

“…41 DNA hairpins with short loops have also been shown to provide selective inhibitors of A3A when the target dC in the loop was changed to dZ-derivatives. [42][43][44] We also demonstrated that dZ and THU as free nucleosides did not inhibit A3 enzymes, which indicates that ssDNA delivers dZ into the active site of A3. 17 In the past, the diazepinone riboside was described as a more powerful inhibitor of CDA than dZ (K i = 25 nM for 1,3diazepin-2-one [31][32][33][34][35] and 2.9 µM for dZ).…”

“…41 DNA hairpins with short loops have also been shown to provide selective inhibitors of A3A when the target dC in the loop was changed to dZ-derivatives. 42–44 We also demonstrated that dZ and THU as free nucleosides did not inhibit A3 enzymes, which indicates that ssDNA delivers dZ into the active site of A3. 17…”

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A

“…Prior studies have shown that linear ssDNA substrates with 2-deoxyzebularine (dZ) and 5-fluoro-dZ (FdZ) in place of the target cytosine are weak inhibitors of A3A (and A3B) [30][31][32] . Additional work by our group and others has shown greater inhibition of A3A in vitro with pre-formed U-shaped, hairpin dZ and FdZ transition-state trapping oligonucleotides [33][34] . Here, we use X-ray crystallography to reveal the first high-resolution structures of wildtype A3A-inhibited complexes and demonstrate the underlying mechanism of inhibition.…”

Protein Dynamics and Enzymatic Catalysis

“…Another area that is potentially available is in the realm of enzyme inhibition. Classical inhibitors have either been unreactive substrate mimics, 152 mechanism-based chemistry inhibitors, 153 or transition state inhibitors. 154 There has been expanded interest in finding ways to inhibit enzymes remote from the active site (to avoid mutational escape from inhibition, for example).…”