4,5-Dihydroxypyrimidine Methyl Carboxylates, Carboxylic Acids, and Carboxamides as Inhibitors of Human Cytomegalovirus pUL89 Endonuclease

Abstract

Human cytomegalovirus (HCMV) terminase complex entails a metal-dependent endonuclease at the C-terminus of pUL89 (pUL89-C). We report herein the design, synthesis, and characterization of dihydroxypyrimidine (DHP) acid ( 14), methyl ester (13), and amide (15) subtypes as inhibitors of HCMV pUL89-C. All analogs synthesized were tested in an endonuclease assay and a thermal shift assay (TSA) and subjected to molecular docking to predict binding affinity. Although analogs inhibiting pUL89-C in the sub-μM range were identified from all three subtypes, acids ( 14) showed better overall potency, substantially larger thermal shift, and considerably better docking scores than esters (13) and amides (15). In the cell-based antiviral assay, six analogs inhibited HCMV with moderate activities (EC 50 = 14.4−22.8 μM). The acid subtype (14) showed good in vitro ADME properties, except for poor permeability. Overall, our data support the DHP acid subtype (14) as a valuable scaffold for developing antivirals targeting HCMV pUL89-C.

8‐Hydroxy‐1,6‐naphthyridine‐7‐carboxamides as Inhibitors of Human Cytomegalovirus pUL89 Endonuclease

“…The samples were incubated in streptavidin coated plates (Pierce Biotechnology, Rockford, IL) at room temperature with gentle shaking for 30 min and then washed three times with 150 μL wash buffer (25 mM Tris, 150 mM NaCl, 0.1 % BSA, and 0.05 % Tween‐20; pH 7.2). Anti‐DIG‐ alkaline phosphatase conjugate antibody (0.15 U/mL) (Roche Applied Sciences, Germany) was added to each well followed by 100 μL of p‐nitrophenylphosphate (1 mg/mL, Sigma‐Aldrich, Saint Louis, MO) as described [39,40] . Absorbance of samples were determined at 405 nm using BioTek Neo 2 plate reader.…”

“…The next day HCMV ADCREGFP virus (obtained from Wade Bresnahan, University of Minnesota) was added at an MOI of 0.01 in DMEM containing 5 % FBS and 1 % P/S for 2 h. After washing with PBS, test compounds were added to each well (final DMSO concentration 0.5 %) and incubated at 37 °C and 5 % CO 2 for 7 days. The cells were lysed to measure GFP fluorescence as an indication of the extent of virus replication as described [39–40] . GFP relative fluorescence units were determined at excitation/emission 495/515 nm in a BioTek Neo2 plate reader.…”

“…Cells were treated the next day with compound in DMSO as described above in the HCMV replication assay except no virus was added. Cellular viability was determined after 7 days using the MTS‐based tetrazolium reduction assay CellTiter 96 Aqueous Non‐Radioactive cell proliferation assay (Promega) as described [40] . Samples were conducted in triplicate and mean values were compared to that for DMSO alone.…”

“…Test compounds (20 μM) were combined with 4 μg of purified pUL89‐C in reaction buffer (5 mM MnCl 2 , 30 mM Tris pH 8 and 50 mM NaCl) for 25 min at room temperature. Sypro Orange Protein Gel Stain (Sigma Aldrich) was added and the assay conducted as previously described [40,42] . All samples were tested in triplicate at a final DMSO concentration of 1 %.…”

“…[20] The RNase Hlike active site structural fold and the metal-dependent catalytic mechanism of pUL89-C are shared by a few other viral metallo enzymatic functions [21][22] we have targeted, including HIV-1 integrase strand transfer (INST), [23][24][25] HIV-1 reverse transcriptaseassociated ribonuclease H (RNase H), [26][27][28][29][30][31][32][33][34] hepatitis B virus (HBV) RNase H, [35] and hepatitis C virus (HCV) NS5B. [36][37] Along the same line, our prior research has identified and characterized a few metal-binding pUL89-C inhibitor types (Figure 2, B): hydroxypyridine carboxylic acid (HPCA, 6), [38][39] dihydroxypyrimidine (DHP) carboxylic acid (7), [40] 6-arylamino-3-hydroxypyrimidine-2,4-dione (HPD-NH, 8), [41] and the 6-arylthio-3-hydroxypyrimidine-2,4-dione (HPD-S, 9). [42] Notably, inhibitor types 6-9 all feature a chelating triad comprising three oxygen atoms for binding two Mn 2 + ions (Figure 2, B, red).…”