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

Abstract

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2′-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A’s preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Molecular mechanism for regulating APOBEC3G DNA editing function by the non-catalytic domain

“…From comparative modeling of our rA3G bound to AA-DNA structure and a previously determined structure of human APOBEC3A bound to a tetraloop DNA hairpin (PDB 8FIK) 62 , we hypothesized that a DNA hairpin substrate with a long stem-length and a 3’overhang (for presenting AA motifs to the non-catalytic CD1 domain) could potentially be edited by rA3G ( Fig. 7a ).…”

“…AA-facilitated DNA editing in hairpin forming sequences performed with the hyperactive variant rA3G R8/P247K/Q317K . (a) Comparative modeling of rA3G bound to AA-DNA (this study), the editing motif CCC-DNA (in pink, modeled from PDB 6BUX 44 ) and a DNA hairpin structure with a tetraloop and a 12-bp stem length (in green, based on PDB 8FIK 62 ). A potential connection path (∼46 Å) between the hairpin DNA (with a 10-bp stem length) and the AA motif is indicated by a black dotted line and labeled as ‘portion of a 3’overhang’.…”

Synthesis of 1,4-azaphosphinine nucleosides and evaluation as inhibitors of human cytidine deaminase and APOBEC3A

“…Nucleoside and polynucleotide (A3) CDA share a universal mechanism of target nucleobase engagement, deamination and inhibition [50,[59][60][61][62][63][64]. We have recently demonstrated the first inhibition of A3A-induced mutagenesis in cells using a DNA hairpin carrying FdZ (IId) instead of the target C in the TTC loop [60].…”

“…Nucleoside and polynucleotide (A3) CDA share a universal mechanism of target nucleobase engagement, deamination and inhibition [50,[59][60][61][62][63][64]. We have recently demonstrated the first inhibition of A3A-induced mutagenesis in cells using a DNA hairpin carrying FdZ (IId) instead of the target C in the TTC loop [60]. To further improve potency of DNA-based inhibitors of A3, more potent inhibitors of cytosine deamination than previously characterised dZ (IIc), FdZ (IId) and diazepinone 2'-deoxyriboside (IIIb) can be used.…”

Development of Allosteric Small Molecule APOBEC3B Inhibitors fromIn SilicoScreening

“…12,13 A well-known cytidine deaminase transition state inhibitor, deoxyzebularine (dZ), was integrated into a ssDNA strand to generate a micromolar inhibitor of A3B, 14,15 and further elaboration of zebularine-based nucleic acid inhibitors have garnered inhibitors with promising biochemical and cellular potency. 16 However, nucleic acid-based analogues of this design require delivery agents or further modifications to enable cellular uptake, limiting utility as probes. Most recently, a number of non-specific A3/AID small molecule inhibitors were published with mid-micromolar inhibitory activity ( Figure S1 ), but these compounds have not been tested for cellular efficacy and are not selective within the A3 family or among the wider APOBEC family.…”