Repurposing N-hydroxy thienopyrimidine-2,4-diones (HtPD) as inhibitors of human cytomegalovirus pUL89 endonuclease: Synthesis and biological characterization

Inhibiting HCMV pUL89-C Endonuclease with Metal-Binding Compounds

“…Further structural evolution by replacing the fused phenyl ring of quinazolinedione analogue 35 with a bioisosteric thiophene ring generated the N-hydroxy thienopyrimidine-2,4-dione (HtPD) chemotype in two isomeric forms: anti-HtPD 36 and syn-HtPD 37 (Figure 6). 13 The SAR studies revealed that majority of HtPD analogues, both anti-isomers 36 and syn-isomers 37, strongly inhibited pUL89-C with IC 50 values typically in the sub-μM range compared to that of 34 and 35 (see Table measurement suggests that poor permeability may have contributed to the relatively poor antiviral potency as compared to the potent pUL89-C inhibition (see Table 4). Also, selected analogues with substantial antiviral activity when evaluated in the biophysical TSA showed significant shift of the pUL89-C melting temperature (36a, ΔT m = 3.5 ± 2.7 °C; 37a, ΔT m = 4.6 ± 0.2 °C) and generated favorable docking scores in the molecular docking studies (36a, XP GScore = −9.3 kcal/mol; 37a, XP GScore = −9.5 kcal/mol), suggesting their engagement with pUL89-C as the protein target.…”

“…69 Inspired by SAR observations of DHP core and similarity of metal dependent catalytic mechanism of pUL89-C to integrase/ RNase H-like viral metalloenzymatic functions, three DHP subtypes were explored as inhibitors of HCMV pUL89-C (Figure 7): methyl carboxylate (41), carboxylic acid (39), and carboxamide (40) (see Table 5). 13,70 The extensive SAR studies based on the functional assay measuring the endonuclease activity of pUL89-C and biophysical TSA showed that the carboxylic acid subtype (39b, pUL89-C IC 50 = 0.6 μM; ΔT m = 5.9 ± 0.5 °C) binds and inhibits pUL89-C better than the ester (41b, pUL89-C 53% inhibition at 5 μM; ΔT m = 2.1 ± 0.5 °C) and amide (40b, pUL89-C IC 50 = 1.0 μM; ΔT m = 1.1 ± 0.4 °C) subtypes. Interestingly, analysis of docking poses of three analogues with 2-thiophene at C-2 of DHP core: acid 39a (XP GScore = −10.7 kcal/mol), ester 41a (XP GScore = −8.6 kcal/ mol), and amide 40a (XP GScore = −7.1 kcal/mol) in active site of HCMV pUL89-C (PDB 6EY7) 38 demonstrated a unique metal-binding mode in which the chelating triad consists of two oxygen atoms and one nitrogen atom rather than the three oxygen atoms observed with previous metal-binding chemotypes (Figure 8).…”

“…Further structural evolution by replacing the fused phenyl ring of quinazoline-dione analogue 35 with a bioisosteric thiophene ring generated the N -hydroxy thienopyrimidine-2,4-dione (HtPD) chemotype in two isomeric forms: anti-HtPD 36 and syn-HtPD 37 (Figure ). The SAR studies revealed that majority of HtPD analogues, both anti-isomers 36 and syn-isomers 37 , strongly inhibited pUL89-C with IC 50 values typically in the sub- μM range compared to that of 34 and 35 (see Table ). A few HtPD analogues, such as 36e (pUL89-C, IC 50 = 1.1 μM; HCMV, EC 50 = 15 μM) and 37f (pUL89-C, IC 50 = 0.60 μM; HCMV, EC 50 = 13 μM), demonstrated significant potencies with low μM EC 50 values in cell-based antiviral assays.…”

“…Recently approved novel drugs include, (B) terminase complex inhibitor letermovir ( 5 ) for HCMV prophylaxis in adult HCMV-seropositive stem cell transplant recipient, and (C) viral kinase inhibitor maribavir ( 6 ) for post-transplant treatment of HCMV. Adapted with permission from ref . Copyright 2022 Elsevier.…”

“…Red square represents cleavage sites on viral DNA genome ( pac sites). Adapted with permission from ref . Copyright 2022 Elsevier.…”