Inhibiting APOBEC3 Activity with Single-Stranded DNA Containing 2′-Deoxyzebularine Analogues

APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2′-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.


A3 enzymes and substrate preferences
In this study we focus on human A3A and the catalytically active C-terminal domains of A3B (A3BCTD) and A3G (A3GCTD) (sequences in Fig. S3). Wild type hA3A and hA3BCTD expressed in human cells were used in fluorescence-based activity assay. Because wild type A3BCTD (wtA3BCTD) does not express well, when purified from E. coli, we used derivatives of A3BCTD (A3BCTD-DM, A3BCTD-QM-ΔL3, and A3BCTD-QM-∆L3-AL1swap). 8 A3BCTD-QM-∆L3-AL1swap is the most active of all A3BCTD listed mutants; A3BCTD-DM is less active but the sequentially closest to wtA3BCTD. A3BCTD-QM-∆L3-AL1swap, A3BCTD-DM, and A3GCTD were used in NMR-based activity and inhibition assays to test dZ-containing oligos. A3BCTD-QM-ΔL3 has very low activity in our NMR assay, and therefore was used in thermal shift assay together with its inactivated mutant A3BCTD-QM-ΔL3-E255A. 9 Additionally, inactivated A3A-E72A, was used to study A3A binding to ssDNA.

Human A3A-E72A expressed in E. coli
For ITC experiments, human APOBEC3A (1-199, Uniprot code P31941) was cloned as the inactive E72A mutant with a His6 C-terminal fusion tag into an expression vector (pETite, Lucigen) and expressed in Escherichia coli BL21 DE3 cells (Hi-Control, Lucigen). The expression medium was supplemented with 100 μM Zn 2+ . Cells were grown in 5 L shake flasks at 37 °C and shortly before induction cooled to 20 °C. Protein expression was induced with 0.25 mM IPTG and protein was expressed at 20 °C overnight. Cells were harvested and resuspended in the following buffer: 25 mM sodium phosphate, pH 7, 500 mM sodium chloride, 5 mM β-mercaptoethanol and 0.2 mM Na2-EDTA or optimized buffer (25 mM sodium phosphate, 500 mM sodium chloride, 5 mM βmercaptoethanol and 0.2 mM Na2-EDTA at pH 6.0 plus 300 mM choline acetate and 0.1 mM LysoFos Choline 12 (Anatrace) as described earlier). 10 Cells were disrupted by sonication or French press, with about twice the yields for the latter method. Lysate was cleared by centrifugation (25 min, 4 °C, 38 000 g) and the supernatant was collected. The protein was purified by metal affinity chromatography. After loading the supernatant, a Ni 2+ -NTA affinity column (BioRad) was washed three times with 10 column volumes of one of the above buffers containing 50 mM imidazole followed by one wash with 200 mM imidazole and eluted with 1 M imidazole. The eluted protein was loaded onto a gel filtration column (Highload 16/600 Superdex 75 pg) and the peak shortly before 100 mL elution volume was collected. The protein was concentrated to about 100 µM using Centricons (Vivaspin 20, 10 kDa MWCO) and aliquots were frozen at -70 °C in the optimized buffer. S10 Fluorescence polarization assays were performed with recombinant (purified from E. coli strain BL21[DE3]), A3A (amino acids 1-195, expressed using the pGEX vector as a GST-fusion) 9 with the catalytic glutamic acid mutated to alanine (E72A) to render the enzyme unable to deaminate substrate. Before use in the fluorescence polarization assay, protein buffer was exchanged from protein storage buffer to FP assay buffer.
The cell culture was harvested via centrifugation and resuspended in 50 mM Tris-HCl (pH 7.4), 0.5 M NaCl, and 5 mM β-mercaptoethanol 50, lysed by sonication and soluble protein was purified through a Ni 2+ -NTA affinity column (BioRad) eluting the protein with 500 mM imidazole. The eluted proteins were loaded through a Superdex 75 10/300 GL column (GE Healthcare) using ÄKTA protein purification system (GE Healthcare) producing a monomeric peak between 90 and 120 mL (see for example Chart S2). The purified proteins were concentrated using Centricons (Vivaspin 20, 10 kDa MWCO) and aliquots were frozen at -80 °C.
A3BCTD -DM was expressed and purified as reported in the recent paper. 8 S11

Human A3GCTD expressed in E. coli
A3GC (191-384, NM_021822, wt) was purified as described. 11 The glutathione Stransferase (GST)-fused A3GCTD was expressed in Escherichia coli BL21(DE3) cells overnight at 17°C. After harvesting, the cells were resuspended in 50 mM sodium phosphate buffer (pH 7.4) and lysed by sonication. After ultracentrifugation at 25,000 g for 10 min, the supernatant was added to glutathione (GSH)-Sepharose, which was subsequently washed. For kinetic analysis, the GST fusion protein was eluted from the Sepharose matrix with 100 mM GSH in phosphate buffer. By using filtration at 4,000 g, the buffer was changed to a solution containing 75 mM sodium phosphate and 75 mM citrate, at pH 5.5. The anisotropy values were then fit to GraphPad Prism log(inhibitor) vs. responsevariable slope (four parameters) function after manual baseline correction to obtain IC50 values for each compound (Table 1, main text, Fig. S2) Where 'Lb' = bound tracer concentration, 'Lo' = total tracer concentration, 'Ro' = total protein concentration, 'Lb' = bound tracer concentration using Equations 2 and 3 to calculate these parameters: We then measured the reliability of the A3A-E72A fluorescence polarization assay by measuring the Z' as recommended for assay validation. 16 Fluorescence polarization assays were performed as described above. Alternating maximum (max), medium

Isothermal titration calorimetry
Desalted unmodified DNA oligonucleotides were purchased (Integrated DNA Technologies) at 1 or 5 µmol synthesis scale and dissolved in one of the buffers described below to give 10 mM solutions.
ITC experiments (Charts S4 -S12) were conducted at 25 °C using a Micro-Cal ITC200 (now Malvern Instruments) isothermal titration calorimeter. A3A-E72A (130 µM in high salt or 33 µM in medium salt buffer) or A3BCTD -QM-∆L3-AL1swap (100 µM, activity assay buffer) was titrated in corresponding buffer. DNA oligonucleotides at 1.6 mM (for A3A-E72A) or 300 µM (for A3BCTD-QM-∆L3-AL1swap) concentration were added in 18 steps at 2.0 µL each (plus a first addition with reduced volume of 0.4 µL to prevent dilution of the DNA in the syringe due to the long wait before the start of the experiment). Oligos and the enzymes were dialyzed against the appropriate buffer. Buffers used are given below: For A3A-E72A: High salt buffer consists of 25 mM sodium phosphate, 500 mM NaCl, 300 mM choline acetate, 5 mM β-mercaptoethanol and 0.2 mM Na2-EDTA, pH 6.0 or medium salt buffer consists of 50 mM MES, 100 mM NaCl, 2.0 mM tris(2carboxyethyl)phosphine, pH 6.0.
Analysis of ITC data was performed with Origin 7 ITC200 software, raw data were fitted to a one-site binding model producing ΔH, ΔS and Ka values (Table S2)   ATTCCdZAATT dZ-modified oligos for direct comparison to substrate (Oligo-3) above and to study A3GCTD inhibition Oligo-8 ATTCCdZ

Thermal Shift Assay
A fluorescence-based thermal shift assay was used to assess binding capability of ssDNA oligonucleotides to A3BCTD proteins, through examination of changes in the proteins thermal stability. Binding assays were conducted using inactive A3BCTD protein constructs, A3BCTD-QM-ΔL3 and A3BCTD-QM-ΔL3 (E255A), where dC substrate is not converted to dU, and to determine if differences in binding occur due to a single amino acid change (E255A) in the protein.
Raw fluorescent intensity data was normalised to percentage, fit to curves, then melting temperature calculation (Tm) was determined at the midpoint (50 %), defined when a protein's melting transition occurs between the folded and unfolded states. Assessment

A3BCTD-QM-ΔL3
S27 of binding of the ssDNA oligonucleotides to the A3BCTD proteins were measured by examining the change in melting temperature (ΔTm) in the presence of oligo compared to absence (buffer) of oligo. Experimental replicates were performed which were analysed using a Q-test for the identification and rejection of outliers based on a 95% confidence interval. Plates were read using BioTek Synergy H1 plate reader with an excitation wavelength at 490 nm and emission at 520 nm. Each experiment was performed in biological duplicate with three technical replicates per condition. Resulting total fluorescence values were reported together with the no-protein low control and protein only high control (no inhibitors). nonspecific inhibitor MN-1 was used as a positive control (Fig. S1).

Kinetic characterisation of A3BCTD-QM-∆L3-AL1swap and A3BCTD -DM
The kinetic characterization of A3BCTD -QM-∆L3-AL1swap and of A3BCTD -DM on dATTTCATTT substrate was conducted using an established one-dimensional proton-NMR (1D 1 H-NMR) based assay. 11 This assay utilizes the naturally abundant proton ( 1 H) isotope nuclei within ssDNA molecule and monitors the real-time deamination of the dC (substrate, dATTTCATTT) to dU (product, dATTTUATTT). Measurements were acquired on a 700-MHz Bruker NMR spectrometer equipped with a 1.7-mm cryoprobe at 298 K. A series of 1 H NMR spectra was recorded of the oligonucleotide substrate 5'-dATTTCATTT at concentrations ranging from 50 µM to 750 µM with 50 nM of A3BCTD -QM-∆L3-AL1swap protein or 2 µM of A3BCTD -DM in a buffer (50 mM citrate-phosphate, 200 mM NaCl, 2 mM β-mercaptoethanol, 200 µM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS); pH 5.5 for A3BCTD -QM-∆L3-AL1swap, pH 7.5 for A3BCTD -DM) containing 10 % deuterium oxide. The H-5 proton doublet signal of the cytosine, which appears between 5.92 to 5.88 ppm, was baselined and integrated (Chart S14, example for A3BCTD -QM-∆L3-AL1swap). A doublet of doublets at 2.57 to 2.39 ppm originating from citrate buffer was used as an internal standard to determine the concentration of substrate converted during the reaction. The integrated signal area was converted to substrate concentration and plotted versus time of the reaction. This plot was then fitted with linear regression to determine the initial speed of the reaction. Charts S15 and S17 show the dependence of speed of the reaction on substrate concentration for A3BCTD -QM-∆L3-AL1swap and A3BCTD -DM, respectively. The double reciprocal plots (Charts S16 and S18) were then fitted with linear regression to determine Km, and kcat using the following formula (showing numbers obtained  Uncertainties of Km and kcat were calculated from regression fit (Chart S16 ) using LINEST function of Excel.

Calculation of inhibition of A3BCTD -QM-∆L3-AL1swap and A3BCTD-DM
by Oligo-9 Based on activity assay described above, measurements of the inhibition of A3BCTD -QM-∆L3-AL1swap and of A3BCTD -DM deaminase activity by dZ-containing oligonucleotide were conducted. A series of 1 H NMR spectra was recorded of the substrate 5'-ATTTCATTT at constant concentration of 350 µM with varying concentrations of the inhibitor 5'-ATTTdZATTT ranging from 5 µM to 100 µM in the presence of 50 nM of A3BCTD-QM-∆L3-AL1swap or of 2 µM of A3BCTD-DM in activity assay buffer as mentioned previously at 298 K. Integration of the H-5 proton doublet signal of the cytosine (between 5.92 to 5.88 ppm) which was then converted to substrate concentration and plotted versus time of the reaction. This plot was then fitted with linear regression to determine the speed of the reaction in the presence of inhibitor. The plot of inverse speed versus inhibitor concentration was then fitted with linear regression to derive the inhibition constant (Ki) using the following formula and Km and kcat values obtained above for A3BCTD -QM-∆L3-AL1swap (numbers shown) and for A3BCTD -DM. Uncertainty of Ki was calculated using error-propagation method.   Figure S4. Deamination of dC in 1mM 5'-dAATTCAAAA by the 100 µM of A3Bctd-QMΔL3 protein over time.