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

Abstract

A DNA hairpin possessing 1,3-diazepin-2-one 2′-deoxyriboside or 5-fluoro-2′-deoxyzebularine in its loop inhibits APOBEC3A in the nM range in vitro.

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 cytidine (C) are weak inhibitors of A3A 40 – 42 , and that these cytidine analogs as free nucleosides are non-inhibitory 42 . Additional work by our group and others has shown greater inhibition of A3A in vitro with U -shaped oligonucleotides containing dZ, FdZ , or 5-methyl-2′-deoxyzebularine transition-state trapping molecules 43 – 45 , which agrees well with biochemical studies comparing linear and hairpin substrates and systematically varying hairpin stems and loops 16 , 26 , 34 , 35 . Here, we use X-ray crystallography to determine high-resolution structures of wildtype A3A/hairpin-inhibited complexes and demonstrate the underlying mechanism of inhibition.…”

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

“…In a manner analogous to that described in reference [ 55 ], we used a 1 H NMR-based assay to test the short oligodeoxynucleotides (ODNs), linear and hairpins, containing individual α- and β-anomers of nucleoside Va as inhibitors of A3. This real-time NMR-based assay is a direct assay: it uses only A3 enzymes and ODNs in a buffer, unlike many fluorescence-based assays where a secondary enzyme and a fluorescently modified oligonucleotide are used [ 72 ].…”

“…The β-anomer of Va was introduced instead of the target dC in the DNA hairpin with TTC loop and tested in the 1 H NMR-based assay monitoring A3A-catalysed deamination of dC hairpin (T(GC) 2 TT C (GC) 2 T, wherein C is deaminated) at a 150 mM salt concentration at pH 7.4. Recently, FdZ ( IId ), 5-methyl-2'-deoxyzebularine and diazepinone 2'-deoxyriboside ( IIIb ) inserted into loops of DNA hairpins have shown selective inhibition of A3A with a half-maximal inhibitory concentration (IC 50 ) and K i in the low-nM range [ 55 , 60 , 76 – 77 ]. Unfortunately, no inhibition of A3A by the DNA hairpin carrying the β-anomer of Va was detected at the concentration used (20 and 100 µM of inhibitor DNA, 1 mM dC hairpin as a substrate, 600 nM of wild-type A3A containing His 6 tag (wtA3A-His 6 ) in 50 mM Na + /K + phosphate buffer, supplemented with 100 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 100 µM sodium trimethylsilylpropanesulfonate (DSS) and 10% D 2 O at pH 4.7).…”

“…We have recently developed the first rationally designed competitive inhibitors of A3 by incorporating 2'-deoxy derivatives of zebularine, i.e., 2'-deoxyzebularine (dZ, IIc ) and 5-fluoro-2'-deoxyzebularine (FdZ, IId , Figure 1B ) [ 54 ] as well as diazepinone 2'-deoxyriboside ( IIIb ) [ 55 ] into DNA fragments. We demonstrated that dZ ( IIc ) does not inhibit A3 enzymes as the free nucleoside but becomes a low-µM inhibitor if it is used in ssDNA instead of the target dC in the recognition motifs of A3A/A3B and A3G [ 54 ].…”

“…The fact that dZ ( IIc ), FdZ ( IId ) and diazepinone 2'-deoxyriboside ( IIIb ) used in the same DNA sequence had a differing inhibitory effect on individual A3 under identical conditions means that the structure of the cytidine analogue determines the inhibitory potential of the inhibitor-containing oligonucleotide [ 55 , 57 ]. This also supports our strategy of using more potent CDA inhibitors in DNA sequences for the development of more powerful A3 inhibitors.…”